Gold nanorod-titanium oxide composite and cosmetic composition for treating acne comprising same

Abstract

The present invention relates to a gold nanorod-titanium oxide composite and a cosmetic composition for treating acne comprising same. The present invention provides a gold nanorod-titanium oxide composite (GNR@TiO2), prepared by coating gold nanorods having a photothermal therapy (PTT) effect with TiO2 having a photodynamic therapy (PDT) effect, and a cosmetic composition for treating acne comprising the gold nanorod-titanium oxide composite. The gold nanorod-titanium oxide composite exhibits superior photothermal effects and reduced in vivo retention compared with conventional microgold (SiO2@Au), and the cosmetic composition comprising the composite provides excellent bactericidal activity against acne-causing bacteria.

Description

Gold nanorod and titanium oxide complex and cosmetic composition for treating acne containing the same

[0001] The present invention relates to a gold nanorod and titanium oxide composite and a cosmetic composition for treating acne containing the same.

[0002] Acne is an inflammatory skin condition that primarily affects the face, neck, back, shoulders, and chest. It is commonly observed in adolescents and significantly impairs their quality of life, leading to psychological withdrawal, depression, and a decline in self-esteem. Acne develops due to the overproduction of male hormones, which causes an increase in keratin in hair follicles. This hyperkeratosis results in pore blockage and increased sebum production. As sebum production increases within the closed pores, acne bacteria proliferate. These bacteria trigger an inflammatory response, causing the follicle walls to rupture and subsequently progressing into pustular acne. If left untreated, pustular acne can negatively impact appearance, leading to redness, hyperpigmentation, or scarring. Therefore, prevention and treatment are necessary to prevent the onset or worsening of acne.

[0003] Acne treatment utilizes near-infrared wavelengths, which have the highest biotransmittance, because the laser must penetrate to the hair follicles. By carefully controlling the laser irradiation time, laser absorption occurs only in the targeted sebaceous glands, thereby enabling their effective destruction.

[0004] One disadvantage of laser treatments is that prolonged use can cause damage to pores due to heat. To address this, photothermal therapy using micro-gold is being utilized. Micro-gold is a composite material (SiO2@Au Core@shell) in which a gold layer is coated on the surface of silica particles. The method involves applying the material to the skin, irradiating it with ultrasound to facilitate penetration, and then applying a laser to destroy sebaceous glands through a photothermal effect. It has been confirmed that applying a laser after micro-gold treatment effectively destroys sebaceous glands, and recovery is rapid due to the short treatment time. However, micro-gold has the disadvantage of requiring additional antibiotic prescriptions because it cannot kill acne bacteria. Furthermore, due to the nature of coating gold on silica particles, the coating is not uniform, which results in a reduced photothermal effect under near-infrared light.

[0005] Accordingly, the inventors investigated a gold nanorod and titanium oxide composite (Gold Nanorod@TiO2Core@shell; GNR@TiO2) to kill acne bacteria simultaneously with photothermal treatment. Since reactive oxygen species (ROS) are generated from the irradiated portion of the gold nanorod and titanium oxide composite when a near-infrared laser is irradiated, thereby killing acne bacteria, the inventors confirmed that the composite can be used for acne treatment by including it in a cosmetic composition, thus completing the present invention.

[0006] The present invention provides a gold nanorod and titanium oxide composite and a cosmetic composition for treating acne containing the same.

[0007] One aspect of the present invention relates to a gold nanorod and titanium oxide composite characterized by a gold nanorod coated with titanium oxide.

[0008] The above gold nanorods may have a negative surface charge.

[0009] The above gold nanorod and titanium oxide composite may have a cell viability of 80% or more at 270 ppm or less.

[0010] The above gold nanorod and titanium oxide composite may have an excellent photothermal effect when irradiated with a laser in the near-infrared region of 700 to 1,400 nm wavelength at a concentration of 50 ppm or more.

[0011] The above gold nanorod and titanium oxide composite may have the effect of killing acne bacteria when irradiated with a laser in the near-infrared region with a wavelength of 700 to 1,400 nm at 100 ± 20 ppm.

[0012] The above gold nanorod and titanium oxide composite may have the effect of killing acne bacteria by generating active oxygen when irradiated with a laser in the near-infrared region of a wavelength of 700 to 1,400 nm.

[0013] The above gold nanorod and titanium oxide composite may not have any gold residue as a result of the bioresidue evaluation in animal skin.

[0014] Another aspect of the present invention is,

[0015] A) A step of preparing a solution containing gold seeds;

[0016] B) A step of manufacturing gold nanorods from the above solution;

[0017] C) a step of replacing the surface charge of the gold nanorod with a negative charge; and

[0018] D) A step of preparing a gold nanorod and titanium oxide composite including the negatively charged gold nanorod and titanium oxide;

[0019] The present invention relates to a method for manufacturing a gold nanorod and titanium oxide composite characterized by including

[0020] In B) above, the gold nanorods and titanium oxide precursors may be mixed in a weight ratio of 1:50 to 150.

[0021] Another aspect of the present invention relates to a cosmetic composition for treating acne containing the gold nanorod and titanium oxide complex.

[0022] The above cosmetic composition for treating acne may contain the gold nanorod and titanium oxide complex at 100 ± 20 ppm.

[0023] The above cosmetic composition for treating acne may reduce the Acne Severity Index (ASI) after 4 weeks of testing.

[0024] In the present invention, a gold nanorod and titanium oxide composite (GNR@TiO2) is synthesized by coating a gold nanorod having a photothermal effect (PTT) with TiO2 having a photodynamic effect (PDT), and a cosmetic composition for treating acne containing the gold nanorod and titanium oxide composite can be provided. The gold nanorod and titanium oxide composite has a superior photothermal effect compared to conventional microgold (SiO2@Au) and has the effect of reducing the amount remaining in the body, and the cosmetic composition for treating acne containing it has the advantage of having an excellent killing effect on acne bacteria.

[0025] Figure 1 is a schematic diagram of the manufacturing process of the gold nanorod and titanium oxide composite of the present invention.

[0026] Figure 2 is a schematic diagram of the energy band gap of the gold nanorod and titanium oxide composite of the present invention.

[0027] Figure 3 is a schematic diagram illustrating the effects of the gold nanorod and titanium oxide composite of the present invention.

[0028] Figure 4 is the result of measuring the zetapotential of a gold nanorod and titanium oxide composite according to an embodiment of the present invention.

[0029] Figure 5 is the FT-IR analysis result of a gold nanorod and titanium oxide composite according to an embodiment of the present invention.

[0030] Figure 6(a) is the result of UV-Vis analysis of a gold nanorod and titanium oxide composite according to an embodiment of the present invention, Figure 6(b) is the result of transmission electron microscopy (TEM) analysis, and Figure 6(c) is a TEM-EDX image.

[0031] FIG. 7 is a schematic diagram of the synthesis of a SiO2@Au composite according to an embodiment of the present invention.

[0032] Figure 8(a) is the result of UV-Vis analysis of a SiO2@Au composite according to an embodiment of the present invention, and Figure 8(b) is the result of transmission electron microscopy (TEM) analysis.

[0033] FIG. 9 is a graph showing the photothermal effects of GNR, gold nanorods and titanium oxide composite, SiO2@Au and microgold according to an embodiment of the present invention.

[0034] Figure 10 shows the results of a cytotoxicity test of a gold nanorod and titanium oxide composite according to an embodiment of the present invention.

[0035] FIG. 11 is a schematic diagram of a photothermal effect experiment on acne bacteria according to an embodiment of the present invention.

[0036] Figure 12 is the result of a photothermal effect experiment on acne bacteria according to an embodiment of the present invention.

[0037] Figure 13 is the result of SYTO9 / Pi double fluorescent staining for acne bacteria according to an embodiment of the present invention.

[0038] Figure 14 is the result of a ROS fluorescence staining experiment on acne bacteria according to an embodiment of the present invention.

[0039] FIG. 15 is a schematic diagram of an animal experiment using a gold nanorod and titanium oxide composite according to an embodiment of the present invention.

[0040] Figure 16 is the result of an animal experiment according to an embodiment of the present invention.

[0041] FIG. 17 is a photograph and a full ingredient list of a cosmetic composition using a gold nanorod and titanium oxide composite according to an embodiment of the present invention.

[0042] Figure 18 is the result of a clinical trial of a cosmetic composition according to an embodiment of the present invention on subjects with acne-prone skin.

[0043] The gold nanorod and titanium oxide composite according to the present invention and the cosmetic composition for treating acne containing the same will be described in detail below. The drawings presented below are provided as examples to ensure that the concept of the present invention is sufficiently conveyed to those skilled in the art. Accordingly, the present invention is not limited to the drawings presented below and may be embodied in other forms, and the drawings presented below may be exaggerated to clarify the concept of the present invention. Unless otherwise defined, technical and scientific terms used hereto have the meaning commonly understood by those skilled in the art to which this invention pertains, and descriptions of known functions and configurations that could unnecessarily obscure the essence of the present invention are omitted in the following description and attached drawings.

[0044]

[0045] The present invention relates to a gold nanorod and titanium oxide composite (GNR@TiO2) and a cosmetic composition for treating acne containing the same, wherein the gold nanorod and titanium oxide composite is characterized by having titanium oxide coated on the gold nanorods.

[0046] The cosmetic composition for treating acne containing the above gold nanorod and titanium oxide complex can destroy sebaceous glands and kill acne bacteria at the same time by utilizing the dual synergy of the photothermal effect of gold nanorods (GNR) and the photodynamic effect caused by the reactive oxygen species (ROS) activity of titanium oxide (TiO2).

[0047] First, the above-mentioned gold nanorods will be explained.

[0048] Metal nanoparticles, such as gold, form Surface Plasmon Resonance (SPR) as free electrons on the surface vibrate through mutual interference with electromagnetic waves from external light. The heat generated by the SPR characteristics of metal nanoparticles can raise the surrounding temperature, and this SPR characteristic occurs more strongly in gold nanorods with a larger aspect ratio compared to spherical gold nanoparticles.

[0049] As the length of gold nanorods increases, the absorption peak at long wavelengths undergoes a red-shift, generally causing strong absorption in the near-infrared region of approximately 700 to 1,200 nm depending on the aspect ratio. When strong near-infrared light is irradiated onto the gold nanorods, free electrons vibrate rapidly along the longitudinal axis of the gold nanorods and collide with gold atoms, converting into heat. This process of photon energy being converted into thermal energy is called the photothermal effect (PTT), and the gold nanorods exhibit excellent photothermal effects within an aspect ratio range of 2 to 7. When irradiated with a laser in the near-infrared region that has excellent biotransmittance, the gold nanorods have the advantage of enabling targeted treatment of acne or sebaceous glands through the photothermal effect.

[0050] The gold nanorods (GNRs) used in the present invention can be synthesized using a seed-mediated growth method. Specifically, a solution containing gold seeds is prepared by adding an aqueous sodium borohydride solution to a solution mixed with an aqueous hexadecyl cetyltrimethylammonium bromide solution and an aqueous gold chloride solution. Then, a gold nanorod solution can be prepared by mixing the aqueous gold chloride solution, hexadecyl cetyltrimethylammonium bromide, and sodium oleate, followed by adding an aqueous silver nitrate solution, which is a synthesis catalyst for gold nanorods, and an aqueous ascorbic acid solution, which is a reducing agent, under stirring. The prepared gold nanorods, after centrifugation and washing with distilled water 2 to 3 times, can be used by subsequently converting their surface charge to a negative charge using sodium dodecyl sulfate to reduce cytotoxicity caused by CTAB.

[0051] Although the volume and concentration of each solution or sample for preparing the Seed solution containing the gold seed are not significantly limited, the Seed solution can preferably be prepared by mixing 1 mL of a 20-30 mM aqueous gold(III) chloride trihydrate (HAuCl4) and 10 mL of 0.1-0.2 M hexadecylcetyltrimethylammonium bromide (Cetrimonium bromide; CTAB), then quickly adding 0.6 mL of a frozen 0.01-0.02 M aqueous sodium borohydride solution and stirring for 3 minutes. It is preferable to age the Seed solution containing the gold seed for at least 30 minutes after preparation; although the time beyond this is not significantly limited, it is preferably aged for 30 minutes to 1 hour before use.

[0052] 0.05–0.06 M CTAB in powder form and 0.008–0.01 M sodium oleate (NaOL) are mixed in 1 L of distilled water, then 20–25 mL of a 25–30 mM aqueous gold chloride solution, 3–5 mL of hydrochloric acid (HCl), 1–2 mL of 0.1–0.2 M ascorbic acid (L-Ascorbic acid; AA), and 10–12 mL of 5–10 mM silver nitrate (AgNO3), which is a gold nanorod synthesis catalyst, are added under stirring, and then 0.8 mL of the aged seed solution can be added under stirring. After 3 hours, 3 to 5 mL of 0.1 to 0.2 M ascorbic acid (AA), a reducing agent, is added and stirred for another 5 to 10 minutes. The final mixture is preferably reacted at 35 °C for at least 12 hours, and it is desirable to wash the final product 2 to 3 times with distilled water using centrifugation to remove excess CTAB.

[0053] Sodium dodecyl sulfate (SDS), an anionic surfactant, can be used to change the surface charge of the washed GNR to a negative charge. 400 mL of 150 ppm gold nanorods from which CTAB has been removed is added to 400 mL of 0.1–0.2 M SDS and stirred for 30 minutes. Afterward, the mixture is centrifuged to remove excess SDS and washed 2–3 times with distilled water to produce gold nanorods with a negatively charged surface as they are coated with SDS.

[0054] In order to coat the gold nanorods prepared in this manner with titanium oxide (TiO2), a titanium oxide precursor solution is prepared and the gold nanorods are mixed to synthesize the gold nanorod and titanium oxide composite of the present invention. The process of coating the gold nanorods, which have a negative surface charge, with titanium oxide by SDS is schematically illustrated in FIG. 1.

[0055] The above titanium oxide precursor solution is a titanium chloride (TiCl₂) as a titanium oxide precursor in distilled water. 3,It may be a mixture containing 10% HCl. It is preferable to adjust the pH of the titanium oxide precursor solution to 2.5 to 3.0 by adding a sodium bicarbonate (NaHCO3) solution. Subsequently, all of the SDS-coated GNR solution is added to the titanium oxide precursor solution and stirred at room temperature for 30 minutes. At this time, the weight ratio of the gold nanorods and the titanium oxide precursor can be mixed in a ratio of 1:50 to 150. The gold nanorod and titanium oxide composite (GNR@TiO2) solution, after synthesis is complete, can be centrifuged, washed 2 to 3 times with ethanol, and then dispersed in final distilled water to produce the gold nanorod and titanium oxide composite (GNR@TiO2).

[0056] The gold nanorod and titanium oxide composite (GNR@TiO2) may have a core-shell structure in which the gold nanorod is the core particle and the titanium oxide is the shell particle. More specifically, the gold nanorod may have an aspect ratio of 2 to 7, and if the aspect ratio is less than 2 or greater than 7, the efficiency of the photothermal effect may decrease. Specifically, the gold nanorod may have an average particle thickness of 10 to 30 nm and a length of 40 to 100 nm. In addition, the titanium oxide, which is the shell particle, may be characterized by having an average thickness of 20 to 80 nm.

[0057] As shown in FIG. 2, the gold nanorod and titanium oxide composite forms a Schottky junction because the Fermi level of the gold nanorod (5.43 eV) in the energy band is higher than the Fermi level of TiO2 (5.10 eV). Due to the Schottky junction, when near-infrared light is irradiated, electrons excited from the gold nanorod move to TiO2, and photodynamic therapy (PDT) occurs due to the electrons that have moved to TiO2. Therefore, as shown in FIG. 3, targeted treatment against acne bacteria is possible based on the generation of reactive oxygen species (ROS) under near-infrared laser irradiation.

[0058] The above gold nanorod and titanium oxide composite can solve the problem of reduced photothermal efficiency caused by the uneven gold layer of microparticles, such as micro-gold particles (SiO2@Au) used in conventional acne treatments, and has the advantage of reducing the burden of having to administer additional antibiotics due to the difficulty of effectively killing acne bacteria. In addition, it can have a superior acne treatment effect compared to existing treatments through synergistic effects with photodynamic therapy using TiO2.

[0059] The above gold nanorod and titanium oxide composite is characterized by a cell viability of 80% or more at 270 ppm or less. Although gold nanorods are one of the gold nanoparticles known to have the least toxicity among metal nanomaterials, nano-sized particles must have low cytotoxicity as they can accumulate in the body. When cytotoxicity was measured by performing the (MTT(3-(4,5)-dimethylthiazo(-z-yl)-3,5-diphenyltetrazoliumromide) Assay on human skin keratinocytes (HaCaT), the above gold nanorod and titanium oxide composite showed a cell viability of 80% or more at 270 ppm or less, thus having the advantage of very low cytotoxicity and excellent biocompatibility.

[0060] The above gold nanorod and titanium oxide composite is characterized by having an excellent photothermal effect when irradiated with a laser in the near-infrared region at a concentration of 50 ppm or higher. The above near-infrared laser may be a laser having a wavelength of 700 to 1,400 nm. When the temperature change is measured upon irradiation with a laser of the above wavelength, conventional microgold exhibits a photothermal effect with a maximum temperature of 55°C at a concentration of 300 ppm or higher, whereas the gold nanorod and titanium oxide composite of the present invention has a similar level of photothermal effect even at 50 ppm, thus having the advantage of having an excellent photothermal effect even at low concentrations.

[0061] The above gold nanorod and titanium oxide composite is characterized by having the effect of killing acne bacteria at 100 ± 20 ppm. When Cutibacterium acnes (C. acnes) is cultured and treated with the above gold nanorod and titanium oxide composite at 100 ± 20 ppm, and then irradiated with a near-infrared laser, the acne bacteria are killed. Specifically, when irradiated with a laser in the near-infrared region with a wavelength of 700 to 1,400 nm, the effect of killing acne bacteria can be exhibited through the generation of reactive oxygen species (ROS), as illustrated in FIG. 3, and this is supported by the observation of ROS fluorescence when the laser is irradiated as shown in FIG. 14.

[0062] In addition, the gold nanorod and titanium oxide composite is characterized by the fact that no gold remains as a result of the in vivo residue evaluation in animal skin. When the gold nanorod and titanium oxide composite was injected and body weight changes were measured during animal in vivo testing, there was no decrease in body weight and no residual amount of gold remained, thus demonstrating low in vivo toxicity. This is also supported by Figure 16 and Table 1, which are the experimental results of confirming in vivo toxicity to be described later.

[0063] The present invention may also provide a method for manufacturing a gold nanorod and titanium oxide composite, characterized by comprising: A) a step of preparing a solution containing a gold seed; B) a step of manufacturing a gold nanorod from the solution; C) a step of substituting the surface charge of the gold nanorod with a negative charge; and D) a step of manufacturing a gold nanorod and titanium oxide composite comprising the gold nanorod substituted with the negative charge and titanium oxide.

[0064] The weight ratio of the gold nanorods and titanium oxide precursors can be mixed in a ratio of 1:50 to 150. Additionally, the components and content of the above manufacturing method are as described above when explaining the gold nanorod and titanium oxide composite, so a redundant description is omitted.

[0065] In addition, the present invention may provide a cosmetic composition for treating acne containing a gold nanorod and titanium oxide complex. The cosmetic composition for treating acne containing the gold nanorod and titanium oxide complex of the present invention may additionally include ingredients conventionally used in cosmetic compositions in addition to the gold nanorod and titanium oxide complex which is the active ingredient, and may include, for example, conventional adjuvants and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances. The formulation of the cosmetic composition may also be formulated as a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, or spray. Furthermore, it may be manufactured as a cosmetic such as a nourishing cream, astringent lotion, softening lotion, lotion, essence, nourishing gel, or massage cream, but is not limited thereto and may be manufactured as any cosmetic formulation conventionally manufactured in the industry.

[0066] Specifically, in the present invention, an ampoule formulation can be prepared using purified water, a gold nanorod and titanium oxide complex (GNR@TiO2), glycerin, butylene glycol, 1,2-hexanediol, arginine, acrylate / C10-30 alkyl acrylate crosspolymer, and carbomer. The cosmetic composition for treating acne may contain 100 ± 20 ppm of the gold nanorod and titanium oxide complex (GNR@TiO2), and preferably 100 ± 10 ppm.

[0067] The above cosmetic composition for treating acne is characterized by a decrease in the Acne Severity Index (ASI) after 4 weeks of testing.

[0068]

[0069] Preferred embodiments of the present invention will be described in detail below. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the content introduced herein is provided to fully convey the concept of the present invention to those skilled in the art, so that it may be thorough and complete.

[0070] Furthermore, unless otherwise defined, all technical and scientific terms have the same meaning as generally understood by one of the art to which the present invention pertains. The terms used in the description herein are merely for the purpose of effectively describing specific embodiments and are not intended to limit the present invention.

[0071]

[0072] <Example 1. Preparation of Gold Nanorod and Titanium Oxide Composite (GNR@TiO2)>

[0073] A) Preparation of solution containing gold seeds: 0.1 mL of 25 mM gold(III) chloride trihydrate (HAuCl4) and 10 mL of 0.1 M hexadecylcetyltrimethylammonium bromide (Cetrimonium bromide; CTAB) were mixed, then 0.6 mL of frozen 0.01 M sodium borohydride (NaBH4) was rapidly added and vortexed for 3 minutes to prepare a seed solution containing gold seeds. The synthesized seed solution was aged at room temperature for at least 30 minutes.

[0074] B) Preparation of gold nanorods: 0.05 M CTAB and 0.008 M sodium oleate (NaOL) were mixed in 1 L of distilled water. Then, 20 mL of 25 mM aqueous gold chloride solution (HAuCl4), 4.2 mL of hydrochloric acid (HCl), 1.6 mL of 0.1 M ascorbic acid (AA), and 10 mL of 10 mM silver nitrate (AgNO3) were added sequentially while stirring, and 0.8 mL of the seed solution from step A) was also added. After 3 hours, 3.2 mL of 0.1 M AA was added and stirred for another 5 minutes. The stirred solution was reacted at 35°C for more than 12 hours, and after centrifugation, the excess CTAB was removed by washing twice with distilled water to obtain gold nanorods.

[0075] C) Preparation of gold nanorods (GNRs) with surface charge substituted to negative charge: 400 mL of 150 ppm gold nanorods from step B) was added to 400 mL of 0.2 M sodium dodecyl sulfate (SDS) and stirred for 30 minutes. Afterward, gold nanorods (GNRs) with surface charge substituted to negative charge were prepared by washing twice with distilled water using centrifugation to wash away excess SDS.

[0076] D) Preparation of gold nanorod and titanium oxide composite (GNR@TiO2) by coating with titanium oxide: 50 mL of TiCl3 (containing 10% HCl), to be used as a precursor for TiO2, was added to 1 L of distilled water, and a 1 M sodium bicarbonate (NaHCO3) solution was added to adjust the pH to 2.5. Subsequently, all of the GNR solution from step C) was added, and the mixture was stirred at room temperature for 30 minutes. The synthesized GNR@TiO2 solution was washed twice with ethanol using centrifugation and dispersed in final distilled water to obtain the gold nanorod and titanium oxide composite (GNR@TiO2).

[0077]

[0078] <Verification of Surface Charge of Gold Nanorods (GNRs)>

[0079] Preparation Example 1: A gold nanorod (GNR) with a surface charge substituted with a negative charge was prepared without performing step D) of Example 1 above.

[0080] Comparative Manufacturing Example 1: A gold nanorod was prepared without performing steps C) and D) of Example 1 above.

[0081]

[0082] For the above Example 1 (GNR@TiO2), Preparation Example 1 (GNR), and Comparative Preparation Example 1, Zeta potential, FT-IR, and UV-vis spectra were analyzed and are shown in FIGS. 4 and 5.

[0083] Zeta potential was measured using Brookhaven 90 plus, FT-IR was measured using Thermo Scientific iD1 Transmission, and UV-vis spectra were analyzed using Shimadzu UV-2600.

[0084]

[0085] Referring to Fig. 4(a), CTAB exhibits a (+) charge as it is a cationic surfactant, SDS exhibits a (-) charge as it is an anionic surfactant, and TiO2 exhibits a weak (-) charge. Looking at Fig. 4(b), Comparative Example 1, which was not treated with SDS, exhibits a (+) charge identical to the surface charge of CTAB, while Example 1 (GNR) exhibits a (-) charge, and Example 1 (GNR@TiO2) also exhibits a (-) charge. Through this, it was confirmed that the surface charges of GNR and GNR@TiO2 are negative.

[0086] Referring to Fig. 5, the FT-IR peaks of Example 1 (GNR@TiO2) are at 634 and 765 cm⁻¹. -1 A peak is observed at 1635 cm⁻¹, which is similar to the SDS peak. -1 And 3361 cm -1In this case, it was confirmed that it is similar to the peaks of the Ti-OH and -OH groups of TiO2.

[0087] The gold nanorod and titanium oxide composite (GNR@TiO2) first reacts the positively charged GNR, mediated by CTAB, with the anionic surfactant SDS to make its surface negatively charged. TiCl3, a precursor of TiO2, is hydrolyzed into TiOH2+ and adsorbed onto the SDS layer, and Ti 3+ Oxidation occurs on the surface of the GNR to form a TiO2 shell. In Fig. 6(a), the absorption spectrum of the GNR shows peaks at 520 nm and 790 nm. Intensity increases in the visible light region after coating with the TiO2 shell; this is thought to be due to the amorphous nature of the TiO2, which causes a rapid increase in scattering, as seen in the TEM image of GNR@TiO2 in Fig. 6(b). Additionally, a red shift in the absorption wavelength in the near-infrared region was observed, which appears to be caused by the coating of TiO2, which has a high refractive index, on the surface of the GNR. In the TEM-EDX image in Fig. 6(c), elements such as Au in the core and Ti and O in the shell were identified. From the above results, it was confirmed that a core-shell structured gold nanorod and titanium oxide composite (GNR@TiO2) was successfully synthesized.

[0088]

[0089] <Verification of Photothermal Effect of Gold Nanorod and Titanium Oxide Composite (GNR@TiO2)>

[0090] Comparative Example 1: To directly prepare SiO2@Au, Au nanoparticles and SiO2 were synthesized. Au particles were prepared by mixing 10 mL of distilled water with 10 mL of 1 mM HAuCl4, stirring at a temperature of about 90 °C or higher, adding 2 mL of 0.35 mmol Trisodium Citrate Dihydrate dissolved in 10 mL of distilled water to the HAuCl4 solution, and stirring until a red light appeared.

[0091] SiO2 particles were synthesized by the sol-gel method. 12 mL of Ammonia solution (NH4OH, 25-29%) was added to 50 mL of anhydrous ethanol, and 0.4 mL of TEOS (Si(OC2H5)4) was slowly added while stirring, and the mixture was stirred at 40 °C for more than 12 hours to prepare a SiO2 mixture. 300 μL of 3-aminopropyltriethoxysilane ((3-Aminopropyl)triethoxysilane; APTES) was added to 10 mL of the SiO2 mixture and stirred at room temperature for 2 hours, then 90 mL of anhydrous ethanol was added and stirred for another hour at 70 °C, after which the mixture was washed by centrifugation and dispersed in distilled water to prepare SiO2-APTES particles. SiO2@Au was prepared by adding 2 mL of SiO2-APTES solution to 20 mL of Au nanoparticles and stirring at room temperature for 1 hour, then adding 1 mL of 3.6 mM Hydroxylamine Hydrochloride solution and stirring for 2 hours, followed by washing via centrifugation, and finally dispersing in distilled water. The synthesis process of the above SiO2@Au is shown in Fig. 7, and the synthesis results are shown in Fig. 8. Looking at Fig. 8(a), it can be observed that as the seed bound to the SiO2 surface grows, the UV-vis absorption wavelength shifts from the visible light wavelength to the near-infrared wavelength. When absorbance was measured, the intensity was measured to be significantly low, which indirectly suggests that the photothermal effect will be reduced. Looking at the TEM image in Fig. 8(b), it can be confirmed that the Au shell is uniformly coated on the SiO2 core.

[0092] Comparative Example 2: Commercially available microgold was prepared.

[0093]

[0094] For the above Example 1 (GNR@TiO2), Preparation Example 1 (GNR), Comparative Example 1 (SiO2@Au (laboratory preparation)), and Comparative Example 2 (micro-gold), the particle concentration was measured using an AAS (MegaA-700FG, Scinco, Korea) to prepare concentrations of 0 ppm, 25 ppm, 50 ppm, and 100 ppm, respectively. While irradiating a 785 nm laser with an intensity of 600 mW using an NIR laser (Chanchun New Industries Optoelectronics Tech, China), the temperature change in real time was measured using an IR-thermo camera (CX320, COX), and the results are shown in Fig. 9.

[0095] Referring to Fig. 9, it was observed that the maximum temperature of Preparation Example 1 (GNR) and Example 1 (GNR@TiO2) was about 55 °C at 50 ppm, whereas the maximum temperature of Comparative Example 1 (SiO2@Au) synthesized directly was about 53 °C at 150 ppm, and the maximum temperature of Comparative Example 2 (Microgold) was about 55 °C at 324 ppm. This appears to be due to the photothermal effect being reduced in Comparative Example 1 (SiO2@Au) because of the characteristic of coating gold on the silica surface. However, seeing that Preparation Example 1 (GNR) and Example 1 (GNR@TiO2) exhibit a similar photothermal effect at 1 / 6 the concentration of Comparative Example 2 (Microgold), it can be seen that they have a superior photothermal effect compared to Microgold.

[0096]

[0097] <Confirmation of Cytotoxicity of Gold Nanorod and Titanium Oxide Composite (GNR@TiO2)>

[0098] The above Example 1 (GNR@TiO2) was prepared, and cytotoxicity was confirmed according to concentration. Human skin keratinocytes (HaCaT, Korean cell line bank (KCLB)) were prepared and cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% heat inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C and 5% CO2. To perform the MTT assay, 20,000 cells / well were aliquoted into a 96-well plate and cultured for 24 hours, after which media treated with the gold nanorod and titanium oxide complex of Example 1 at concentrations of 0 ppm, 30 ppm, 60 ppm, 90 ppm, 120 ppm, 150 ppm, 180 ppm, 210 ppm, 240 ppm, 270 ppm, and 300 ppm were incubated for 12 hours.

[0099] After replacing the medium in the 96-well plate, 50 μL of 0.005 g / mL MTT solution was added to each well and incubated for 3 hours. Subsequently, the supernatant was removed, and 500 μL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the generated formazan. The absorbance at 540 nm was then measured using a microplate spectrophotometer (BioTek), and the cell viability was calculated and is shown in Figure 10.

[0100] Referring to Figure 10, the cell viability was approximately 78% even at a high concentration of 300 ppm, and especially at a concentration of 270 ppm or less, the cell viability was over 80%, indicating that the cytotoxicity of the gold nanorod and titanium oxide composite (GNR@TiO2) is very low.

[0101]

[0102] <Confirmation of Photothermal Effect on Acne Bacteria>

[0103] The above Example 1 (GNR@TiO2) and Preparation Example 1 (GNR) were prepared to compare the photothermal effects on acne bacteria. Acne bacteria (Cutibacterium acnes (C. acnes), Korean Collection for Type Cultures (KCTC)) were streaked onto Reinforced Clostridial Medium (RCM) solid medium and cultured for at least 24 hours under conditions of 36 °C and 5% CO2. Colonies were collected from the streaked RCM solid medium and pre-cultured in RCM liquid medium. The bacteria pre-cultured in RCM liquid medium were OD 600 =0.1 (10 8 It was adjusted to CFU / mL and diluted 1,000-fold. Using the diluted bacteria, GNR, GNR@TiO2, and RCM liquid media, the bacterial concentration was set to 1 x 10⁶ 4 After adjusting to CFU / mL, 1 mL of the solution was placed in a 48-well plate as shown in Fig. 11, and a 780 nm NIR laser was irradiated at an intensity of 600 mW for 20 minutes. After laser irradiation, 100 μL of the solution and 100 μL of the solution diluted 100-fold were spread onto an RCM solid medium, followed by spreading and incubation for more than 24 hours.

[0104] Acne bacteria concentration 1 x 10 in Fig. 12(a) 4 A photograph of CFU / mL was shown, and in Fig. 12(b), an acne bacteria concentration of 1 x 10 2 A photograph of CFU / mL is shown, and in Fig. 12(c), a bacterial concentration of 1 x 10 2 The number of colonies in CFU / mL was measured and graphed.

[0105] Referring to Fig. 12(a), the bacterial concentration is 1 x 10 4When plated on an RCM solid medium at CFU / mL, it can be seen that the GNR@TiO2 100 ppm bacteria irradiated with a laser were completely killed. Figure 12(b) is a 100-fold dilution of (a) to calculate the number of colonies, and when the number of colonies was measured based on (b), looking at the graph in Figure 12(c), it can be seen that after laser irradiation, approximately 50% of the bacteria in Preparation Example 1 (GNR) were killed, while 100% of the bacteria in Example 1 (GNR@TiO2) were killed. The temperature during laser irradiation was the same for Preparation Example 1 (GNR) and Example 1 (GNR@TiO2), and from this, it can be seen that the complete killing of acne bacteria in Example 1 (GNR@TiO2) was due to the photothermal effect of the gold nanorod and titanium oxide composite, rather than the photothermal effect of gold.

[0106]

[0107] <Acne Bacteria Killing Effect Confirmed>

[0108] The above Example 1 (GNR@TiO2) and Preparation Example 1 (GNR) were prepared to compare the killing effect on acne bacteria. SYTO9 / Propidium iodide (Pi) double staining was performed to confirm, under a microscope, the degree of killing of acne bacteria after laser irradiation. In 1 mL of solution (RCM medium, an acne bacteria culture medium), 100 ppm of Example 1 (GNR@TiO2) and 1 x 10⁶ acne bacteria were added. 9 After adding CFU and irradiating with a laser in the same manner as the photothermal experiment, the culture medium was removed by centrifugation, and the acne bacteria were suspended in 50 μL of phosphate-buffered saline (PBS, pH=7.0). The suspension was then mixed with 50 μL of Working Solution (30 μL of 2.5 mM SYTO9, 60 μL of Pi, 30 μL of PBS) under dark conditions and stained for 20 minutes. After staining, 5 μL of the solution was sprayed onto a slide glass and covered with a cover glass. SYTO9 was observed at 470 nm and Pi at 525 nm using a confocal microscope, and the results are shown in Figure 13.

[0109] SYTO9 is membrane-permeable and passes through the cell membrane to stain nucleic acids and emit green fluorescence, staining both living and dead bacteria, whereas Pi is membrane-impermeable and stains nucleic acids of bacteria with damaged cell membranes and emits red fluorescence.

[0110] As shown in FIG. 13, when looking at images before and after laser irradiation after treating acne bacteria with 100 ppm of Preparation Example 1 (GNR) and Example 1 (GNR@TiO2), in the case of the Control that was not treated with Preparation Example 1 (GNR) or Example 1 (GNR@TiO2), there is no significant difference before and after laser irradiation as shown in FIG. 13(a). On the other hand, in Preparation Example 1 (GNR), Pi was partially stained after laser irradiation as shown in FIG. 13(b), and in Example 1 (GNR@TiO2), Pi was completely stained as shown in FIG. 13(c), confirming that the cell membrane of the acne bacteria was damaged and killed.

[0111]

[0112] <Confirmation of Reactive Oxygen Species (ROS) Generation by Acne Bacteria>

[0113] 100 ppm of Example 1 (GNR@TiO2) and 1 x 10 of acne bacteria in 1 mL of solution (RCM medium, an acne bacteria culture medium). 9 Acne bacteria, after adding CFU and irradiating with a laser in the same manner as the photothermal experiment, with the culture medium removed by centrifugation, were suspended in 80 μL of PBS and stained in 20 μL of 2',7'-dichlorodihydrofluorescein diacetate (H2CFH-DA) for 20 minutes under dark conditions. After staining, 5 μL of the solution was spread onto a slide glass, covered with a cover glass, and observed under a fluorescence microscope at 470 nm. The results are shown in Fig. 14.

[0114] H2DCF-DA is hydrolyzed by intracellular esterase to become non-fluorescent DCF-H, and has the principle of being oxidized to fluorescent DCF in the presence of intracellular ROS.

[0115] As shown in Fig. 14, when viewing images before and after laser irradiation after treating acne bacteria with 100 ppm of Preparation Example 1 (GNR) and Example 1 (GNR@TiO2), no ROS fluorescence was observed in the Control (which was not treated with Preparation Example 1 (GNR) or Example 1 (GNR@TiO2)) and Preparation Example 1 (GNR) before and after laser irradiation, while ROS fluorescence was observed in Example 1 (GNR@TiO2) after laser irradiation, it can be confirmed that irradiating Example 1 (GNR@TiO2) with a laser causes ROS activity and kills acne bacteria.

[0116]

[0117] <in vivo 생체 독성 확인>

[0118] The above Example 1 (GNR@TiO2) was prepared, and its in vivo toxicity was tested through animal experiments (Daegu Gyeongbuk Advanced Medical Industry Promotion Foundation Preclinical Center) as shown in Fig. 15. For the acne animal model, 6 mice were administered to the group receiving Example 1 (GNR@TiO2), and 6 mice were administered to the control group. C. acnes strain 1 x 10 8After centrifuging and settling the CFU, it was suspended in 20 μL of PBS and injected into the skin of the right dorsal side of rats. Strain transplantation was performed under isoflurane inhalation anesthesia, and after confirming that the rats had recovered from anesthesia, they were reared normally. The GNR@TiO2 dosage was administered uniformly to all individuals at a single dose of 50 μL at 100 ppm, followed by irradiation with a 780 nm NIR laser at an intensity of 450 mW for 10 minutes, 3 hours later. Changes in rat body weight over 33 days are shown in Figure 16. Blood, liver, and skin tissue samples from the lesion site were collected from all animals via necropsy. The collected blood, liver, and skin tissue samples were analyzed for Au concentration using an ICP-OES (Inductively Coupled Plasma Optical Emission Spectrometer) at the Joint Laboratory of Kumoh National Institute of Technology, and the results are shown in Table 1.

[0119] As shown in Figure 16, the body weight of mice in each group showed a steady increase during the experimental period, and it was confirmed that no weight loss occurred due to the injection of Example 1 (GNR@TiO2). Through this data, it can be seen that the biotoxicity of the gold nanorod and titanium oxide composite is not significant.

[0120] As shown in Table 1 below, when the element Au was analyzed by ICP-OES in blood, liver, and skin tissue samples collected during the autopsy, the element Au was not detected. This result indicates that even if the gold nanorod and titanium oxide composite is injected into the skin, there is no or very little residual amount in the body.

[0121] Au Concentration Average (ppm) Untreated Group (Control) Example 1 (GNR@TiO2) Impacted Group Blood - 0.0005 - 0.001 Liver - 0.0005 - 0.001 Skin - 0.0005 - 0.001

[0122]

[0123] <Clinical Trial on Subjects with Acne-Prone Skin>

[0124] Example 1 (GNR@TiO2) was prepared and tested on human acne-prone skin. The human application test was conducted through the Korean Institute of Dermatological Sciences and involved 11 patients with acne-prone skin (5 men, 6 women, average age 27). Using Example 1 (GNR@TiO2), an ampoule formulation was prepared using purified water, 100 ppm of Example 1 (GNR@TiO2), glycerin, butylene glycol, 1,2-hexanediol, arginine, acrylate / C10-30 alkyl acrylate crosspolymer, and carbomer, as shown in Fig. 17. After cleansing the face of the acne patient, the Example 1 (GNR@TiO2) ampoule was applied once to the lesion area, and PDT treatment was performed using an NIR laser after 10 to 20 minutes. After taking facial photographs of the subjects before the test, and 2 and 4 weeks after the test, two experts evaluated the Acne Severity Index (ASI), and the results are shown in Fig. 18.

[0125] Referring to Fig. 18(a), in the group treated with the Example 1 (GNR@TiO2) ampoule and PDT during a total test period of 4 weeks, it was confirmed that the Acne Severity Index (ASI) decreased to a statistically significant level after 4 weeks compared to before the test, and as shown in Fig. 18(b), the reduction in acne could also be visually confirmed.

[0126] Through the above experiments, it can be seen that the gold nanorod and titanium oxide composite of the present invention is highly useful as a cosmetic composition for treating acne, and in particular, that its effect is enhanced under conditions of near-infrared laser irradiation, thereby enabling a more pioneering method for treating acne.

Claims

1. A gold nanorod and titanium oxide composite characterized by gold nanorods coated with titanium oxide.

2. In Paragraph 1, The above gold nanorod and titanium oxide composite is characterized by the gold nanorod having a negative surface charge.

3. In Paragraph 1, The gold nanorod and titanium oxide composite is characterized by having a cell viability of 80% or more at 270 ppm or less.

4. In Paragraph 1, The gold nanorod and titanium oxide composite is characterized by having an excellent photothermal effect when irradiated with a laser in the near-infrared region of a wavelength of 700 to 1,400 nm at a concentration of 50 ppm or more.

5. In Paragraph 1, The gold nanorod and titanium oxide composite is characterized by having the effect of killing acne bacteria when irradiated with a laser in the near-infrared region of a wavelength of 700 to 1,400 nm at 100 ± 20 ppm.

6. In Paragraph 1, The gold nanorod and titanium oxide composite is characterized by having the effect of killing acne bacteria by generating active oxygen when irradiated with a laser in the near-infrared region of a wavelength of 700 to 1,400 nm.

7. In Paragraph 1, The above gold nanorod and titanium oxide composite is characterized by the fact that no gold remains as a result of an in vivo residue evaluation in animal skin. 8.A) A step of preparing a solution containing gold seeds; B) A step of manufacturing gold nanorods from the above solution; C) a step of replacing the surface charge of the gold nanorod with a negative charge; and D) A step of preparing a gold nanorod and titanium oxide composite including the negatively charged gold nanorod and titanium oxide precursor; A method for manufacturing a gold nanorod and titanium oxide composite characterized by including 9. In Paragraph 8, A method for manufacturing a gold nanorod and titanium oxide composite, characterized in that the gold nanorod and titanium oxide precursor in B) above are mixed in a weight ratio of 1:50 to 150.

10. A cosmetic composition for treating acne containing a gold nanorod and titanium oxide complex according to any one of claims 1 to 7.

11. In Paragraph 10, The above cosmetic composition for treating acne is characterized by containing the above gold nanorod and titanium oxide complex at 100 ± 20 ppm.

12. In Paragraph 10, The above-described cosmetic composition for treating acne is characterized by a decrease in the Acne Severity Index (ASI) after 4 weeks of testing.

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