KR102064672B1 - Reducing agent-assisted excessive galvanic replacement mediated seed-mediated synthesis of porous alloy nanostructures and porous alloy nanostructures using the same - Google Patents

Abstract

The present invention, in the production process of silver nanoplates (solution), by introducing one or more metal ions selected from gold, platinum, palladium under conditions containing a specific additive to the porous alloy nanostructures in one-pot reaction It is simple and convenient to prepare, but it is possible to prevent fragmentation and aggregation of the porous alloy nanostructures by specific additives, and to adjust the amount of metal ions added, the type and concentration of additives, The present invention relates to a method of manufacturing a porous alloy nanostructure that can arbitrarily control the shape of the nanostructure.

Description

REDUCING AGENT-ASSISTED EXCESSIVE GALVANIC REPLACEMENT MEDIATED SEED-MEDIATED SYNTHESIS OF POROUS ALLOY NANOSTRUCTURES AND POROUS ALLOY NANOSTRUCTURES USING THE SAME }

The present invention relates to a method for producing a porous alloy nanostructures through a reducing agent-assisted excess galvanic substitution reaction, and to a porous alloy nanostructures prepared through the same.

Research into colloidal nanoparticle design and synthesis technology that can be used in the field has been continued. Meanwhile, silver (Ag) nanoplates, which are anisotropic nanoparticles, have been studied for a long time because of their two-dimensional planar structure and unique physicochemical properties. Today, in order to collectively modulate their local surface plasmon resonance (LSPR) properties, attempts to modify the edge length, thickness and shape of these nanoplates, as well as postsynthesis (such as chemical etching, photo-etching and elemental substitution reactions) Research activities to produce silver nanoplates with various characteristics using postsynthetic transformation techniques are also actively conducted.

Meanwhile, the galvanic substitution reaction, which is widely known as the post-synthesis modification technique, is a kind of redox reaction in a solution phase based on the standard reduction potential difference between the two elements, and the silver (Ag) and copper (Cu) through the galvanic substitution reaction. When adding substituted ions such as gold (Au), platinum (Pt) and palladium (Pd) to a sacrificial template such as, and cobalt (Co), the hollow nanoplate or Modified nanostructures such as nanoskeleton can be prepared.

Meanwhile, in the case of gold nanoplates manufactured through galvanic substitution, electromagnetic waves can be absorbed by local surface plasmon resonance, and the absorbed electromagnetic waves collectively vibrate conductive electrons in the metal film to generate thermal energy. . Accordingly, since the elevated temperature is sufficient to irreversibly denature the tertiary or quaternary structure of the protein, the gold nanoplates are known to be used for photothermal therapy (or heat therapy) for cancer cells.

Meanwhile, J. Kimling et al. In the prior art measured the absorption spectrum of 5 to 120 nm of gold nanoparticles reduced with ascorbic acid and stabilized with sodium citrate. Meanwhile, Brian G. Prevo et al. Published a study in which gold nanoparticles were stabilized with sodium citrate and reduced with sodium borohydride to alloy silver nanoparticles for photothermal treatment. have.

However, in the case of the above methods, the synthesis process of gold nanoplates is not only complicated, but also fragmentation and agglomeration phenomena occur in manufacturing nanostructures. In addition, the prepared nanostructures had low photothermal conversion efficiency and did not have sufficient volume-to-surface ratios required for the carrier loading, thus making them unsuitable for use in gene-fever complex therapy.

On the other hand, unlike the gold nanoparticles described above, platinum (Pt) or palladium (Pd), which is a noble metal-based nanoparticle, shows high cytotoxicity, high synthesis cost, and local surface plasmon resonance in the ultraviolet region (UV). (LSPR) is generally thought to be limited and has not been applied as a common chemotherapy delivery vehicle.

Republic of Korea Patent Publication No. 10-2011-0044668 (2011.04.29.)

An object of the present invention, in the process of preparing a silver nanoplate solution, by introducing one or more metal ions selected from gold, platinum and palladium under conditions containing a specific additive to convert the porous alloy nanostructures into a one-pot reaction It is simple and convenient to prepare, but it is possible to prevent fragmentation and aggregation of the porous alloy nanostructures by specific additives, and to adjust the amount of metal ions added, the type and concentration of additives, The present invention provides a method of manufacturing a porous alloy nanostructure that can arbitrarily control the shape of the nanostructure.

In addition, it is prepared according to the above method, has a high absorbance for near-infrared (NIR) laser irradiation in the 800 ~ 900nm wavelength range, showing excellent light-to-conversion effect, and easy to load the carrier due to the high volume-to-surface ratio to cancer cells To provide a porous alloy nanostructures having selective gene-fever complex therapeutic ability.

In order to achieve the above object, in the present specification, a silver nanoplate solution is prepared from a silver nitrate (AgNO ₃ ) solution, and the silver nanoplate solution includes gold (Au), platinum (Pt), and palladium (Pd). Injecting at least one metal ion selected from the group to perform a galvanic substitution reaction, to prepare a porous alloy nanostructure substituted with metal ions, the step is trisodium citrate (Na ₃ Cit), poly ( Vinylpyrrolidone) and L- ascorbic acid provides a method for producing a porous alloy nanostructures carried out in a one-pot reaction under conditions containing one or more additives selected from the group consisting of.

In the present specification, at least one metal selected from the group consisting of gold (Au), platinum (Pt), and palladium (Pd); And silver (Ag); Provided is a porous alloy nanostructure comprising an alloy of and exhibiting a photothermal conversion effect by near-infrared (NIR) laser irradiation in the 800 to 900 nm wavelength region.

According to the present invention, in the process of preparing the silver nanoplate solution, the porous alloy nanostructures are simple-to-one-pot reactions by injecting one or more metal ions selected from gold, platinum and palladium under conditions containing specific additives. And conveniently prepared, and prevents fragmentation and aggregation of the porous alloy nanostructures by specific additives, and controls the amount of metal ions added, the type and concentration of additives, The shape can be controlled arbitrarily.

In addition, the nanostructures prepared according to the above method have high absorbance for near-infrared (NIR) laser irradiation in the wavelength range of 800 to 900 nm, exhibit excellent light-to-conversion effect, and easy to load carrier due to high volume-to-surface ratio. Has selective gene-fever complex therapeutic ability against cancer cells.

In particular, the porous alloy nanostructures comprising an alloy of palladium (Pd) and silver (Ag) prepared according to the above method are useful for photothermal conversion, therapeutic gene loading / release, cytotoxicity and in vitro complex anticancer. As it exhibits outstanding all-round superiority in treatment, it is suitable for use in the field of anti-cancer therapeutic delivery and biopharmaceuticals.

1 shows TEM images and LSPR spectra of silver (Ag) nanoplates scaled according to one embodiment of the invention.
Figure 2 shows the disappearance spectrum of the TEM image and UV-Vis NIR region of the porous alloy nanostructure obtained according to an embodiment of the present invention.
Figure 3 shows the analysis of the characteristics of the porous alloy nanostructures obtained in accordance with an embodiment of the present invention by XRD and XPS.
4 shows a wide-scan XPS of pAuNPs, pPtNPs, and pPdNPs films on a silicon wafer obtained in accordance with one embodiment of the present invention.
FIG. 5 shows HR-TEM and HAADF-STEM / EDS images of (a) pAuNPs, (b) pPtNPs and (c) pPdNPs, which are porous alloy nanostructures obtained in accordance with one embodiment of the present invention.
6 shows EDS spectra of pAuNPs on silicon wafers obtained in accordance with one embodiment of the present invention.
7 shows EDS spectra of pPtNPs on silicon wafers obtained in accordance with one embodiment of the present invention.
8 shows EDS spectra of pPdNPs on the resulting silicon wafer, in accordance with an embodiment of the present invention.
Figure 9 shows the element mapping through the SEM image of the porous alloy nanostructures obtained in accordance with an embodiment of the present invention.
Figure 10 shows the cytotoxicity test results using the porous alloy nanostructures obtained in accordance with an embodiment of the present invention.
FIG. 11 shows a temperature increase as a result of irradiating 4 W / cm ² 808 nm NIR near infrared light on the porous alloy nanostructure obtained according to one embodiment of the present invention.
Figure 12 shows the results of measuring the DLS and ζ potential of the TAT bonded porous alloy nanostructures obtained in accordance with an embodiment of the present invention.
FIG. 13 shows the loading / release profile of FAM fluorescently labeled and thiolated DMAzyme on porous alloy nanostructures obtained in accordance with one embodiment of the present invention.
Figure 14 shows a quantitative gene-fever complex treatment efficiency analysis of the porous alloy nanostructures obtained according to one embodiment of the present invention.

Hereinafter, a porous alloy nanostructure manufacturing method and a porous alloy nanostructure manufactured according to a specific embodiment of the present invention will be described in detail.

Method of manufacturing porous alloy nanostructures (pMNPs)

As described above, according to an embodiment of the present invention, a silver nanoplate solution is prepared from a silver nitrate (AgNO ₃ ) solution, and gold (Au), platinum (Pt), and palladium (Pd) are contained in the silver nanoplate solution. Injecting at least one metal ion selected from the group consisting of performing a galvanic substitution reaction, to prepare a porous alloy nanostructure substituted with metal ions, the step is trisodium citrate (Na ₃ Cit), poly (Vinylpyrrolidone) and L- ascorbic acid may be provided a method for producing a porous alloy nanostructures carried out in a one-pot reaction under conditions containing at least one additive selected from the group consisting of.

The inventors of the present invention provide a porous alloy nano in a one-pot reaction when an excessive amount of one or more metal ions selected from gold, platinum, and palladium is added under a condition in which a specific additive is included in the process of preparing a silver nanoplate solution. When the structure can be produced simply and conveniently, the fragmentation and aggregation of the porous alloy nanostructures are prevented by specific additives, and the amount of metal ions added, the type and concentration of the additives are controlled. , It was confirmed through experiments that the shape of the nanostructure to be manufactured can be arbitrarily controlled. In addition, the porous alloy nanostructures prepared according to the above method have high absorbance for near-infrared (NIR) laser irradiation in the 800-900 nm wavelength region, and exhibit excellent light-to-conversion effect, and due to the high volume-to-surface ratio, the carrier loading is high. It was confirmed through experiments that it has a selective gene-fever complex therapeutic ability to cancer cells to facilitate the present invention was completed. In particular, the porous alloy nanostructures comprising an alloy of palladium (Pd) and silver (Ag) prepared according to the above method are useful for photothermal conversion, therapeutic gene loading / release, cytotoxicity and in vitro complex anticancer. As it exhibits outstanding all-round superiority in treatment, it is suitable for use in the field of anti-cancer therapeutic delivery and biopharmaceuticals.

According to one embodiment of the present invention, the process of preparing silver nanoplates (solutions) may be by a general method in the art, and specifically, poly (vinylpyrrolidone) (PVP) in silver nitrate (AgNO ₃ ) solution. ), By adding an additive such as trisodium citrate (Na ₃ Cit), hydrogen peroxide, sodium borohydride, etc., silver nanoseeds can be prepared first, and then scaled through seed-based growth to be prepared as silver nanoplates (solutions). have. As an example, according to an embodiment of the present invention, the silver nanoplate may be one sized by seed-based growth (see FIG. 1), and local surface plasmon resonance (LSPR) may be red shifted to the near infrared region. . For reference, the scale bar of FIG. 1 is 100 nm.

According to one embodiment of the present invention, the metal ion introduced into the silver nanoplate solution receives electrons from silver atoms on the surface of the silver nanoplate by galvanic substitution reaction, which is a redox reaction on a solution based on a standard reduction potential difference. It serves to perform the plating. According to one embodiment of the present invention, the metal ion which is a substituted ion may be an ion of at least one metal selected from the group consisting of gold (Au), platinum (Pt), and palladium (Pd), and specifically palladium (Pd) ions. For example, the metal ion, which is a substitution ion, may be added to the silver nanoplate (solution) in the form of chloroacetic acid (AuCl ₄ ⁻ ), chloroplatinic acid (PtCl ₄ ^2- ), or palladium chloride (PdCl ₄ ^2- ).

On the other hand, according to an embodiment of the present invention, the metal ion in the step, may be added in 1 to 20% based on the total solution volume of silver (Ag) nanoplate. In the galvanic substitution reaction according to an embodiment of the present invention, excessive addition of metal ions, which are substituted ions, may be made. When the metal ions are excessively substituted, an alloy nanoplate having a porosity is produced by inducing a balance change between substitution and reduction rates. Can be.

On the other hand, by adjusting the amount (concentration) of the metal ions that are substituted ions, it is possible to arbitrarily control the shape of the porous nanostructure to be produced. The porous alloy nanostructures prepared according to one embodiment of the present invention may have one or more shapes selected from the group consisting of nanoplates, hollow nanoplates, nanoskeletons and uneven nanoplates.

Specifically, when the metal ion, which is a substitution ion, is a gold ion, structures such as nanoplates, hollow nanoplates, nanoskeletons, and uneven nanoplates are all possible, but in the case of platinum ions or palladium ions, porous nanoplates or uneven It can have only the same shape as the nanoplate.

On the other hand, according to an embodiment of the present invention, the step is under conditions containing at least one additive selected from the group consisting of trisodium citrate (Na ₃ Cit), poly (vinylpyrrolidone) and L- ascorbic acid It can be carried out in a one-pot reaction. Specifically, it may be carried out under the conditions in which all three additives described above are included.

In particular, when manufacturing the alloy nanostructures according to the prior art, first to prepare a silver nanoplate, the silver nanoplate is prepared and then used or assumed to be an additive component used as impurities to remove or purify it. Next, when the silver nanoplate is transferred to the alloy nanoplate, the reaction is carried out by adding additional components. In the present invention, after the silver nanoplate is prepared, metal ions are added without additional purification or additive component removal. It is differentiated from the prior art in that the alloy nanostructure is manufactured by a one-pot reaction, and thus, it is advantageous in terms of economy and process convenience compared to the prior art, and furthermore, the porous alloy nanostructure is carried out by performing the reaction under a specific additive combination. It is possible to prevent the fragmentation (fragmentation) and aggregation (aggregation) of the phenomenon, and to arbitrarily control the shape of the nanostructure to be manufactured.

In addition, in general, when manufacturing a modified nanostructure using an excessive galvanic substitution reaction, there was a problem that severe fragmentation (aggregation) and aggregation (aggregation) of the nanostructure occurs. Under the additive conditions according to an embodiment of the present invention, especially when the reaction is performed under the conditions including all three additives described above, fragmentation and aggregation of the nanostructures are effectively performed by synergistic and complementary effects of the additives. Can be prevented. In addition, in addition to the amount (concentration) of the metal ions described above, the type and concentration of the additive may also act as a factor enabling the nanostructure shape control.

Trisodium citrate (Na ₃ Cit), one of the additives according to an embodiment of the present invention serves as a surface-capping stabilizer, may contribute to the dispersion of nanomaterials. In the absence of trisodium citrate, there may be a problem that silver chloride (AgCl) formed as a by-product of the galvanic substitution reaction is concentrated and aggregated on the surface of small nanoparticle fragments produced. On the other hand, when trisodium citrate is included as an additive, the above problems can be effectively prevented by the additional stabilization effect caused by the adsorption behavior of trisodium citrate (Na ₃ Cit) on the surface of the nanomaterial.

Poly (vinylpyrrolidone) (PVP), one of the additives according to an embodiment of the present invention, is to prevent fragmentation of the nanostructures in an excessive galvanic substitution reaction. At the time, the initial structure of the nanoplate is not maintained, and there is a problem of fragmentation into small debris.

One of the additives according to an embodiment of the present invention, L-ascorbic acid (L-AA), as a secondary reducing agent, may prevent fragmentation in the galvanic substitution reaction and may serve to secondaryly grow the nanostructure. . In the absence of L-AA, the galvanic substitution causes the nanostructure to deform more quickly by the addition of excess gold chloride (AuCl ₄ ⁻ ), chloroplatinic acid (PtCl ₄ ^2- ) or palladium chloride (PdCl ₄ ^2- ), More fragmentation is shown.

According to one embodiment of the present invention, the above step may be carried out under conditions including trisodium citrate (Na ₃ Cit), poly (vinylpyrrolidone) and L-ascorbic acid as additives, for example trisodium citrate (Na ₃ Cit), poly (vinylpyrrolidone), and L-ascorbic acid may be included in concentrations of 0.15 mM, 30 mM, and 1 mM, respectively, based on 40 ml of the silver (Ag) nanoplate solution. When included in the concentration effectively prevents fragmentation (fragmentation) and aggregation (aggregation) of the nanostructures. In addition, the nanostructures produced have a high volume-to-surface ratio to facilitate carrier loading and thus have a selective gene-fever complex therapeutic ability against cancer cells.

Porous Alloy Nanostructures

On the other hand, the porous alloy nanostructures according to the embodiment of the present invention prepared according to the above method, gold (Au), platinum (Pt) and palladium (Pd) selected from the group consisting of at least one metal; And silver (Ag); It contains an alloy of and exhibits a light-heat conversion effect by near-infrared (NIR) laser irradiation in the 800-900 nm wavelength region.

On the other hand, as described above, the porous alloy nanostructure according to an embodiment of the present invention, the shape is controlled by adjusting the amount of one or more factors selected from the group consisting of the amount of metal ions, the type of additives and the concentration of additives It may be prepared as, specifically, may have a shape of one or more selected from the group consisting of nanoplates, hollow nanoplates, nanoskeleton and uneven nanoplate.

On the other hand, the photothermal conversion effect of the porous alloy nanostructures prepared according to one embodiment of the present invention seems to start from the incident light absorption and electron vibration for the kinetic energy transfer through the electron-phonon interaction. The main absorption wavelength of the porous alloy nanostructures (pAuNPs, pPtNPs or pPdNPs) prepared according to one embodiment of the present invention is the entire UV-Vis-NIR region, and has excellent absorbance even in the 800-900 nm region, especially 800- It is suitable for the medical application mainly utilizing the 900nm wavelength, which shows an excellent light-heat conversion effect compared to the conventional spherical nanoparticles.

On the other hand, in the case of having such a shape, it is easy to load the carrier due to the high volume to surface ratio. Accordingly, the porous alloy nanostructure according to an embodiment of the present invention is one or more carriers selected from DNA-based enzymes (DNAzyme) and TAT cell penetrating peptides at one or more positions selected from inside and the pores of the porous alloy nanostructures. Can be loaded. As such, when one or more selected from DNA-based enzymes (DNAzyme) and TAT cell penetrating peptides are loaded at one or more positions selected from inside and the pores of the porous alloy nanostructures, a selective gene-generating complex for cancer cells It may have therapeutic power.

Meanwhile, as a result of analyzing through ICP-MS according to an embodiment of the present invention, when the porous alloy nanostructure is an alloy of gold (Au) and silver (Ag), the ratio of gold / silver is about 3.64. In another example, an alloy of platinum (Pt) and silver (Ag) may have a platinum / silver ratio of about 2.53. In another example, an alloy of palladium (Pd) and silver (Ag) may be used. In the case of the palladium / silver ratio was confirmed to correspond to 1.96.

In particular, among the prepared porous alloy nanostructures, the porous alloy nanostructures including an alloy of palladium (Pd) and silver (Ag) are photothermal conversion, therapeutic gene loading / release, cytotoxicity and in vitro (in vitro) ) As it shows outstanding all-round superiority in complex chemotherapy, it is suitable for use in chemotherapy delivery means and biopharmaceutical field.

Carrier Loading for Porous Alloy Nanostructures

On the other hand, in the porous alloy nanostructures manufacturing method according to an embodiment of the present invention, the porous alloy nanostructures prepared by the above-described step is loaded with one or more carriers selected from DNA-based enzymes (DNAzyme) and TAT cell penetrating peptides It may further comprise a.

On the other hand, the porous alloy nanostructures prepared according to one embodiment of the present invention, when compared to the conventional spherical gold nanoparticles commonly used in the field of drug delivery and treatment, near infrared (NIR) laser irradiation in the 800 ~ 900nm wavelength region It has a high absorbance against and shows excellent light-to-conversion effect, and its high volume-to-surface ratio makes it easy to load carriers and thus has a selective gene-fever complex therapeutic ability for cancer cells. Can be.

As described above, during the preparation process of the silver nanoplate solution, the porous alloy nanostructures are converted into a one-pot reaction by introducing one or more metal ions selected from gold, platinum, and palladium under conditions including specific additives. Prepared simply and conveniently, the specific additives to prevent the fragmentation (fragmentation) and aggregation (aggregation) phenomenon of the porous alloy nanostructures, and to control the amount of metal ions added, the type and concentration of additives, nanostructures produced The shape of can be controlled arbitrarily. In addition, the nanostructures prepared according to the above method have high absorbance for near-infrared (NIR) laser irradiation in the wavelength range of 800 to 900 nm, exhibit excellent light-to-conversion effect, and easy to load carrier due to high volume-to-surface ratio. As it has a selective gene-fever complex therapeutic ability against cancer cells, it is expected to be used more effectively in the field of drug delivery and treatment.

Example

As the invention allows for various changes and numerous modifications, particular embodiments will be illustrated and described in detail below. However, this is not intended to limit the present invention to the specific disclosed form, it should be understood to include all modifications, equivalents, and substitutes included in the spirit and scope of the present invention.

Example 1 Preparation of Porous Alloy Nanoplates

Preparation of Silver (Ag) Nanoseeds

250 ml of 10 mM silver nitrate (AgNO ₃ ), 300 µl of 30 mM trisodium citrate (Na ₃ Cit), 1.5 ml of poly (vinylpyrrolidone) (Mw = 29 kDa) and 24.75 ml of deionized water were added to a glass vial ( vial), 60 μl of 30% hydrogen peroxide was added, followed by gentle stirring for uniform mixing. 100 mM sodium borohydride was added to the mixture to give a pale yellow color. Then, after 3 hours of reaction time, it was confirmed that no further color change was progressed after the change to transparent, dark yellow, orange, and finally purple, and proceeded to the next growth reaction step without further purification.

Synthesis of Silver (Ag) Nanoplates by Seed Growth Method

To 10 ml of the seed solution was added 0.125 ml of 75 mM trisodium citrate (Na ₃ Cit) and 0.375 ml of 100 mM L-ascorbic acid. Next, a growth solution was prepared with 20 ml of 1 mM silver nitrate (AgNO ₃ ), 0.125 ml of 100 mM citric acid, and 10 µl of 75 mM trisodium citrate (Na ₃ Cit). 5 ml of growth solution was added to the silver nanoseed solution at a rate of 0.1 ml per second. Nanoseed color changed to dark blue with growth solution addition. After 10 minutes of further incubation, the silver nanoplate solution was converted into pMNPs through galvanic substitution without further purification (M = Au, Pt and Pd).

Synthesis of Porous Alloy Nanoplates (pMNPs) by Galvanic Substitution

The prepared silver (Ag) nanoplatelet solution was diluted 5-fold with an appropriate amount of deionized water. It was mixed ^- (, PtCl ₄ ^2- and PdCl ₄ ^2- AuCl ₄₎ solution of 20 v / v% direct injection and turned over several times the dilution is (Ag) 1 mM substituted ions to nano-plate solution. The solution color changed at different rates such as pAuNP (light blue, purple and dark blue within 30 minutes), pPtNP (light blue to brown black within 2 hours) and pPdNP (pale blue, brown and brown black within 1 hour). After incubation for 2 hours at room temperature without interference, the synthesized pMNPs were purified by centrifugation at 9,000 rpm for 10 minutes and washed three times with deionized water. Finally, pMNPs were redispersed in deionized water as the volume of silver (Ag) nanoplatelets before dilution used for galvanic substitution and expressed as 10 eq.

Specifically, according to the TEM image, porous alloy nanostructures (pAuNPs) including an alloy of gold / silver obtained using gold ions, and porous alloy nanos including an alloy of platinum / silver obtained using platinum ions. The structures (pPtNPs) and the porous alloy nanostructures (pPdNPs) comprising palladium / silver alloys (Alloy) obtained using palladium ions each have a well preserved appearance with a porous structure and a rough surface morphology with a transverse diameter of 80 to 100 nm. It can be seen that the low magnification image supports the uniformity of the prepared pMNPs (M = Au, Pt or Pd) (see Fig. 2 (a) to (c)). On the other hand, based on this specific nanostructure, pAuNPs show peaks of 650 to 750 nm in the LSPR spectrum, pPtNPs and pPdNPs show maximum absorption in the UV region, and gradually decrease toward 1,100 nm, but the overall absorption efficiency is high. Appeared (see FIG. 2 (d)). The incident photons caused the oscillation of the surface plasmon, resulting in a photothermal conversion effect. Thus, pPdNPs and pPtNPs exhibiting high absorbance over the entire wavelength can be utilized in the UV-Vis-NIR region. In addition, the near-infrared source used in general biomedical applications is a diode-based laser and has a relatively wide wavelength range of 400 to 1,000 nm, so Vis-NIR nanos exhibit a broad absorption spectrum even when the maximum emission wavelength is regarded as 808 nm. The particles may be advantageous in terms of light heat conversion.

Experiment 1 Characteristics of pMNPs

Images of synthesized pMNPs were obtained using an energy filtration transmission electron microscope LIBRA 120 (Carl Zeiss, Germany), JEM-2100F HR (JEOL Ltd., Japan), and a field-emission scanning electron microscope AURIGA (Carl Zeiss, Germany). X-ray diffraction was measured by D8-Advance (Bruker Miller Co., USA). X-ray photoelectron spectroscopy was measured with AXIS-HIs (KRATOS, UK). UV-Vis spectrophotometer Lambda 465 (PerkinElmer, USA) and SynergyMx (Biotek, UK) were used to obtain UV-Vis-NIR absorption spectra. Dynamic light scattering and -potential were measured by Zetasizer Nano ZS (Malvern, UK). Fluorescence was measured with a spectrophotometer FP-8300 (Jasco Inc., USA). 808 nm NIR irradiation was performed with OCLA (Soodogroup Co., Korea), a surgical laser accessory. Cell images were taken using an In-cell analyzer 2000 (GE healthcare, USA), a Ti reverse fluorescence microscope (Nikon Co., Japan) and a CoolSNAP cf charge coupled device (CCD) camera (Photometrics, USA).

In order to perform a specific analysis of the alloy form composition observed in the form of pieces in the TEM, X-ray diffraction (XRD) analysis for further confirmation of the specific composition and crystallinity of the synthesized pMNPs was carried out using all the elements (Ag, Au, Pt and Pd) has a plane-centered cubic (fcc) structure, and the crystal plane is clearly observed. In general, inorganic element exchange through galvanic substitution results in the formation of alloy compositions, and from EDS analysis, pMNPs are in the form of Au-Ag (pAuNPs), Pt-Ag (pPtNPs) and Pd-Ag (pPdNPs). It can be seen that the amount of the introduced element is composed of the dominant alloy. Three pMNPs diffraction peaks are located between simulated broad peak positions of the pure element, which means alloy phase formation. Si (002) peaks due to Si wafer based thin film XRD analysis are detected (see FIG. 3), and further confirmation of surface elemental composition analysis using X-ray photoelectron spectroscopy (XPS) for pMNPs, the dominant substitution The binding energy information for each of the metal element species is given by Au 4f _5/2 (87eV) and pAuNPs combined with silver (Ag) signals of Ag 3d _3/2 (373e V) and Ag 3d _5/2 (367eV). Au 4f _7/2 (83.3eV), Pt 4f _5/2 (73.75eV) for pPtNPs, Pt 4f _7/2 (70.25eV), Pd 3d _3/2 (339.9eV) for PPdNPs, Pd3d _5/2 (334.6 eV) (see FIGS. 3 and 4). Comprehensive X-ray analysis confirmed that the porous nanoplates formed through galvanic substitution consisted of coexistence of substituted metal elements and silver (Ag) templates.

In addition, for confirming observations from previous X-ray characterization, proximity analysis was performed on energy dispersive spectroscopy (EDS) mapping (see FIG. 5), high-resolution (HR) TEM, and high-angle annular dark-field (HAADF) STEM. We focused on a broad analysis of the overall characterization by scanning electron microscopy (SEM) with single nanoparticles and EDS. Nanoplate morphology and composition were further characterized by field emission scanning electron microscopy (SEM) using energy dispersion spectroscopy (EDS). According to the SEM image, 20 nm thick nanoplate morphology is clearly observed from partially aggregated samples formed by evaporation. The EDS spectrum clearly shows the coexistence of small amounts of silver (Ag) remaining in the alloy form with substituted elements for pMNPs (see FIGS. 6-8). Although the background mapping image could not be clearly identified due to the background noise, it was possible to distinguish the presence of substitution elements (Au, Pt and Pd) and template sacrificial elements (Ag) in the corresponding pMNPs (see FIG. 9). We found that pMNPs consisted of alloys of substitution elements (M = Au, Pt or Pd) and template elements (Ag) that produce pAuAgNPs, pPtAgNPs and pPdAgNPs, but for simplicity, we use the nano Plates are referred to as pAuNPs, pPtNPs, pPdNPs, respectively.

Example 2: Preparation of TAT Peptide, FAM-Dz Loaded Porous Alloy Nanoplates

Loading of TAT Peptides and FAM-Dz on pMNPs

20 μL of 1 μM TAT peptide and 15 μL of 1 μM FAM-Dz-Sh (FDz) were added to 1 eq. The solution was placed in 1 ml of pMNPs (M = Au, Pt, Pd). The mixture was incubated for 12 h in a horizontal shaker at 180 rpm in the dark. Unbound TAT peptide was removed by centrifugation at 7,000 rpm for 15 minutes and washed with deionized water at least three times. Finally, TAT peptide loaded pMNPs were redispersed in 1 ml of 1 × PBS. Calculation of the loaded FDz was made by fluorescence intensity measurement of unbound FAM (λ _ex = 495 nm, λ _em = 520 nm) in the supernatant after centrifugation.

FAM-Dz Release Profile

To monitor the release of loaded FDz under cytoplasmic-mimicked conditions, FDz / TAT-pMNPs and FDz-pMNPs (M = Au, Pt and Pd) were treated with 1 × PBS (pH) with or without 2 mM glutathione at room temperature. 7.4) dispersed in solution. Observe every 0, 1, 2, 3, 6, 9, 12, 18 and 24 hours, centrifuge FDz / TAT-pMNPs and FDz-pMNPs at 7,000 rpm for 15 minutes to release and release FDz-pMNPs nanocomposites The amount of FDz added was determined by fluorescence intensity measurement.

Experiment 2: Photothermal Conversion Characteristics

Temperature rise measurement

To assess the temperature rise by photothermal conversion, 1 equivalent of 1 ml pMNPs and control 1 × PBS were placed in a 2 ml tube. A 808 nm NIR diode laser was irradiated to each sample at an intensity of 4 W / cm ² for 2 minutes, and the temperature change was measured with a digital thermometer every 30 seconds.

Photothermal cancer cell resection

To examine hyperthermic cancer cell resection by photothermal conversion, a 12-well plate NS3 replicon seeded with 1 equivalent TAT-pMNPs in 1X PBS at a confluency of 80,000 cell / well Treatment with Huh7 cells. In a 37 ° C. condition CO- ₂ incubator at 5% humidity, after 6 hours of incubation, residual TAT-pMNPs were removed and washed twice with 1 × PBS and then replaced with serum containing medium. Next, the cells were irradiated with an 808 nm near infrared diode laser having an intensity of 4 W / cm ² for 5 minutes at ambient conditions and incubated for an additional 12 hours. After incubation, 500 μl of bound live / dead cell staining solution (2 μM Calcein AM and 4 μM Ethid-1 in D-PBS) was added to each well and incubated for 20 minutes for successful staining. Fluorescence microscopy was used to obtain fluorescence images of the cells.

Experiment 3 Measurement of In Vitro Treatment Efficiency

Cell culture

Human liver cancer cell line Huh7 containing NS3 hepatitis virus RNA was supplemented with 4.5 g / L D- with 10% FBS, 100 units / mL penicillin, 100 mg / mL streptomycin and 500 μg / mL G418 (neomycin). Growing in DMEM containing glucose. The cells were grown in a humidified 5% CO ₂ incubator at 37 ° C.

Cytotoxicity measurement

MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) powder was dissolved in 1 × PBS at a concentration of 5 mg / ml and a 0.2 μm pore size sterile syringe filter Filtered through. The stock solution was stored at 4 ° C. NS3 replicon Huh7 cells were seeded at a density of 10,000 cells per well of 96-well culture plates with 100 μl of growth medium (about 50-70% confluency).

To compare drug delivery efficiency based on TAT conjugated nanoparticle groups and non-conjugated nanoparticle groups, cells were treated with free FDz, FDz-pMNPs and FDZ / TAT-pMNPs in serum-free medium and 37 ° C. for 6 hours. Incubated at. Cells were then washed twice with 1 × PBS, replaced with serum containing medium and incubated at 37 ° C. for 12 hours. The cells were then rinsed twice with 1 × PBS. 100 μl of serum free medium was added to the cells in MTT at 0.5 mg / ml concentration and incubated for 2 hours until purple was developed to detect metabolic active cells. Discard the medium, rinse the cells once with 1X PBS to remove the remaining MTT. 100 μL of dimethylsulfoxide (DMSO) was then added to each well to solubilize the water insoluble formazan salt. The optical density of each well in the plate was measured at 560 nm. Three times the mean and standard deviation were calculated and plotted.

To identify further therapeutic applications, cytotoxicity assays of pAuNPs, pPtNPs and pPdNPs on Huh-7 human liver cancer cells infected with hepatitis C virus (HCV) nonstructural protein 3 (NS3) replication (NS3-Huh7 cells) Was performed. Because of the difficulty in quantifying the molar concentrations of pMNPs (M = Au, Pt or Pd), the concentration criterion is the disappearance of silver (Ag) nanoplates under the assumption that the silver (Ag) nanoplates are not lost by fragmentation during the substitution reaction. Is set to. For quantitative in vitro analysis, the concentration of pMNPs synthesized via galvanic substitution was set to 1 equivalent. Compared to the untreated control, all three types of pMNPs were 0.5eq. It showed negligible cytotoxicity up to concentration (95.53%, 96.98% and 98.65% viability in the presence of 1 equivalent of pAuNPs, pPtNPs and pPdNPs, respectively). For AgNPs, clear cytotoxicity was 0.125 eq. It started appearing above concentration. 0.5eq. At higher concentrations, dramatic cytotoxicity was observed with similar trends in all cases of Au, Pt, and Pd. Based on the UV-Vis-NIR absorption using the highest non-toxic concentration (1 eq.), We found 0.5 eq. In the presence of pMNPs, the following in vitro experiments were performed. The observed cytotoxicity was thought to be the result of the hydrodynamic diameter and surface charge homogeneity of the pMNPs via reducing agent assisted excess galvanic substitution. In addition, at least in terms of cytotoxicity, pPdNPs and pPtNPs may have new biomedical applications that can replace existing gold nanomaterials (see FIG. 10 (a)).

Genetic Heat Complex Therapeutic Efficiency

To investigate the synergistic effect of NIR radiation with pMNPs derivative-based chemotherapy, NS3-Huh7 cells were treated with free FDz, TAT-pMNPs and TAT / FDz-pMNPs in serum-free medium and incubated at 37 ° C. for 6 hours. It was. Cells were then rinsed twice with 1 × PBS and replaced with serum containing medium. Next, the cells were irradiated with an 808 nm near infrared laser having an intensity of 4 W / cm ² for 2 minutes at ambient conditions and incubated at 37 ° C. for 12 hours. Thereafter, the cells were rinsed twice with 1 × PBS. 100 μl of serum free was added to the cells in MTT at 0.5 mg / ml concentration and incubated for 2 hours until purple was expressed to detect metabolic active cells. The medium was discarded and the cells were rinsed with IX PBS. 100 μl DMSO was then added to each well to solubilize the formazan salt insoluble in water. The optical density of each well in the plate was measured at 560 nm. Three times the mean and standard deviation were calculated and plotted.

Next, the temperature rise induced by the photothermal conversion effect was measured when irradiated with a 808 nm NIR diode laser at a nontoxic concentration of pMNPs of 0.1 eq. After applying 4 W / cm ² NIR laser irradiation for 120 seconds, pPdNPs showed the best photothermal conversion efficiency (T _f = 52.3 ° C., ΔT = 31.0 ° C.), followed by pPtNPs (T _f = 48.5 ° C., ΔT =). 27.2) ° C), pAuNPs (T _f = 45.8 ° C, ΔT = 24.5 ° C) and AgNP (T _f = 37.0 ° C, ΔT = 15.8 ° C). Compared to the negligible temperature rise of 1X phosphate buffered saline (PBS) induced by the radiant heat of the light source (T _f = 22.5 ° C., ΔT = 1.2 ° C.), all pMNPs have sufficient photothermal conversion to achieve high temperature photothermal treatment. The efficiency was shown (see FIG. 10 (b) and FIG. 11).

On the other hand, in order to efficiently insert pMNPs into NS3-Huh7 cells, we describe thiols derived from human immunodeficiency virus CGG YGRKKKRRQRRR (underlined letters represent essential sequences of TAT peptides, bold letters represent thiol-containing sequences for conjugation). The oxidized cell permeable peptide (TAT) was conjugated to the metal-thiol affinity pMNPs surface. Successful surface loading of TAT peptides was characterized by dynamic light scattering (DLS) and ζ-potential measurements. pAuNPs and pPdNPs were successfully treated with TAT peptides by increasing the hydrodynamic diameters (Δd = 12.26 and 8.9 nm for pAuNPs and pPdNPs, respectively) and ζ-potentials (Δ ζ = 3.73 and 5.97 mV for pAuNPs and pPdNPs, respectively). Modified together, where the TAT loading efficiency of pPtNPs was significantly lower (Δd = 0.01 nm and Δζ = 1.24 mV). Since the hydraulic diameters were calculated from the transformation diffusion coefficients through the Stocks-Einstein equation with inherent problems due to approximations such as spheres, the DLS data of porous nanoplates can be ambiguous. However, the ζ − potential increases from positively charged TAT-peptide modifications, supporting the higher loading efficiency decreased in the order of pPdNPs, pAuNPs and pPtNPs (see FIG. 12).

Next, NS3-Huh7 cells were treated with 1 equivalent of prepared TAT-pMNPs and incubated for several hours to facilitate insertion, followed by a 808 nm NIR diode laser (4 W / w) to induce photothermal conversion based hot cell ablation. cm ² , 5 minutes). Fluorescence microscopy after survival / kill staining with calcein AM and EthiD-1 revealed hot cell death in the designed area (see FIG. 10 (b)).

Specifically, before verifying the therapeutic effect through gene delivery, we analyzed gene loading and release profiles for pMNPs using fluorescently labeled end-thiolated DNAzyme (FAM-Dz-SH) targeting HCV NS3 mRNA. Was performed. Briefly, we added 150 pmol FAM-Dz-SH to 1 eq. Was mixed in the dark with pMNPs, and time dependent loading was observed by measuring the fluorescence of unbound FAM-Dz-SH in the supernatant after centrifugation. According to 24 hours of observation, rapid FAM-Dz-SH loading was achieved within 3 hours of mixing, followed by saturation after incubation for 12 hours at room temperature. The loading efficiency of FAM-Dz-SH was shown in the order pPdNPs (92.3 pmol)> pAuNPs (72.5 pmol)> pPtNPs (28.2 pmol)> AgNPs (7.9 pmol) (see FIG. 13 (a)). Release of FAM-Dz to pMNPs was investigated by exposing FAM-Dz / pMNPs to cytoplasmic mimetics containing glutathione (GSH) at a concentration of 2 mM in 1 × PBS for reduction of metal-thiol bonds. In the absence of GSH, the amount of FAM-Dz released was negligible at the error range level for all three pMNPs (about 2% release). However, in the presence of 2 mM GSH, FAM-Dz release during incubation for 24 hours was found to be FAM-Dz-pPdNP (83.6%)> FAM-Dz / pAuNP (67.9%)> FAM-Dz-pPtNP (63.4%)> FAM -Dz-AgNP (19.4%) was shown in the order (see Fig. 13 (c) and (e)).

Differences in thiolated cargo loading loads (pPdNP>pAuNP>pPtNP> AgNPs) during TAT peptide loading, FAM-Dz loading and FAM-Dz / TAT double loading are more important for chemical factors than physical factors such as diameter, shape and composition It suggests that. As a result of TEM, SEM, and DLS analysis, three pMNPs showed similar areas and ζ-potential values were similar. In addition, EDS and XRD analysis clearly showed the same fcc crystal plane with alloy composition, suggesting that the chemical affinity between major metal elements (Au, Pt and Pd) and thiols could be a key factor. Bond dissociation energy and binding of Ag-S (216.7 ± 14.6 kJ / mol), Au-S (379.5 ± 6.3 kJ / mol), Pd-S (398 kJ / mol) and Pt-S (262 ± 10 kJ / mol) According to the enthalpy, the binding affinity between metal elements and thiols decreased in the order of Pd>Au>Pt> Ag. Because little previous work has been done on the affinity and binding of thiols to Pt, this data was inferred from known thermodynamics, but showed the same trend as the experimental results.

For efficient gene delivery, pMNPs conjugated with cell penetrating peptide TAT and therapeutic gene FAM-Dz were prepared by co-mixing. Because the cargo strategy for both cargoes is the same, the amount of FAM-Dz loaded on NPs is reduced. For quantitative verification, load / emission profiling was again performed for kinetic confirmation. Based on the same loading strategy of metal-thiol affinity adsorption, the FAM-Dz loading in the case of double cargo loading was 73.3% (pAuNPs), 68.7% (pPdNPs), 38.7% (relative to the FAM-Dz loading profile alone). pPtNPs) and 85.8% (AgNPs) tended to decrease (FIG. 6B). In the FAM-Dz release profile of the FAM-Dz / TAT double load, under conditions mimicking the intracellular environment (2 mM GSH), 6.52% (pAuNPs), 7.92% (pPtNPs) and 5.02 compared to the FAM-Dz single load test % (pPdNPs) was reduced respectively. For AgNPs, a small increase in the margin of error was observed in the emission profile. However, specific release efficiencies were observed compared to 0 mM GSH conditions (~ 2% FAM-Dz release), and the FAM-Dz / TAT double loading effect was considered sufficient when considering improved cell permeability for effective gene delivery (FIG. 13 (d) and (f)).

Finally, MTT cell viability assays were used to assess the efficacy of gene-heat combination therapy of cancer in NS3-Huh7 cells. We compared relative cell viability based on untreated control conditions. NIR irradiation alone (100.1% survival), pMNPs treatment (pPdNPs 99.2%, pAuNPs 101.7%, pPtNPs 101.3%) and TAT-pMNP treatment without treatment genes (pPdNPs 100.6%, pAuNPs 101.7%, pPtNPs 100.1%) No significant cell death was observed. Even when cells were treated with 150 pmol of free FAM-Dz alone, no cell death was observed (104.0% survival). Also, FAM-Dz-pMNPs (99.7% for pPdNPs, 100.7% for pAuNPs, 101.6% for pPtNPs) and NIR-based photothermal therapy (100.2% for pPdNPs, 101.6% for pAuNPs, 99.6% for pPtNPs) without the cell penetrating peptide TAT. There was no therapeutic effect in the presence of pMNPs. We also confirmed that TAT peptides play an important role in the cellular internalization of pMNPs. Gene therapy efficiencies with double cargo loaded nanocomposite FAM-Dz / TAT-pMNPs (56.2% for pPdNPs, 65.7% for pAuNPs and 88.7% for pPtNPs) were significantly improved. In addition, in the photothermal treatment group using TAT-pMNPs, we compared the photothermal conversion-based temperature rise of each type of pMNPs in 1X PBS with a trend (pPdNPs 61.0%, pAuNPs 68.8%, pPtNPs 64.5%). It was confirmed that the match. As a result of the final gene-thermal combined treatment efficiency, the best efficiencies such as pPdNPs (0.1% viability), pAuNPs (16.7% survival) and pPtNPs (25.9% viability) were obtained. In a series of experiments, we used a shorter irradiation time (2 minutes) than photothermal conversion based temperature rise and region specific hyperthermic cell death experiments (5 minutes), which was the optimal condition chosen to observe the synergistic effect of the combination treatment. (See FIG. 14 (a)).

To further confirm FAM-Dz induced delivery and cancer cell death, fluorescence microscopy images were obtained after staining Hoechst 33342 (cell nucleus, blue) and EthiD-1 (cell death, red). The other experimental group did not show any fluorescence signal, but the nanocomposite treatment group conjugated with the TAT peptide showed the corresponding fluorescence signal (see FIG. 14 (b)).

Claims (9)

A silver nanoplate solution is prepared from a silver nitrate (AgNO ₃ ) solution, and galvanic is prepared by adding one or more metal ions selected from the group consisting of gold (Au), platinum (Pt), and palladium (Pd) into the silver nanoplate solution. Performing a substitution reaction to prepare a porous alloy nanostructure substituted with metal ions,
The step is a method of producing a porous alloy nanostructures are carried out by a one-pot reaction by the addition of trisodium citrate (Na ₃ Cit), poly (vinylpyrrolidone) and L- ascorbic acid. The method of claim 1,
And loading at least one carrier selected from DNA-based enzymes (DNAzyme) and TAT cell penetrating peptides into the porous alloy nanostructures. The method of claim 1,
In the step, at least one metal ion selected from the group consisting of gold (Au), platinum (Pt), and palladium (Pd) is added at 1 to 20% based on the total solution volume of silver (Ag) nanoplates. Method for producing a porous alloy nanostructures. The method of claim 1,
Method for producing a porous alloy nanostructures that controls the shape of the nanostructures produced by adjusting the amount of the metal ions, the type of additives and at least one selected from the group consisting of additive concentration. A porous alloy nanostructure prepared according to the method of claim 1,
At least one metal selected from the group consisting of gold (Au), platinum (Pt), and palladium (Pd); And silver (Ag); A porous alloy nanostructure comprising an alloy of and having a photothermal-conversion effect and selective gene-heating composite therapeutic ability against cancer cells by near infrared (NIR) laser irradiation in the 800-900 nm wavelength region. The method of claim 5,
The porous alloy nanostructures are porous alloy nanostructures including an alloy of palladium (Pd) and silver (Ag). The method of claim 5,
The porous alloy nanostructures are porous alloy nanostructures having one or more shapes selected from the group consisting of nanoplates, hollow nanoplates, nanoskeletons and uneven nanoplates. The method of claim 5,
Porous alloy nanostructures are loaded with one or more carriers selected from DNA-based enzymes (DNAzyme) and TAT cell penetrating peptides at one or more positions selected from inside and the pores of the porous alloy nanostructures. delete

Images