Pharmaceutical composition for preventing or treating cancer comprising upconversion nanoparticles

Abstract

The present invention relates to a nanoplatform comprising upconversion nanoparticles having a core-shell structure comprising a core made of NaYF4:Yb, Tm; and a shell made of NaYF4:Nd surrounding the core, and to a pharmaceutical composition or food composition comprising the same.

Description

Technology Field

[0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of cancer comprising upconversion nanoparticles. Background Technology

[0003] Cryoablation and cryospamming are cryotherapy approaches for various human cancers involving the lethal repeated freezing and thawing of cancer cells.

[0004] Despite the long history of this treatment practice, it is insufficient to completely block cancer progression. Recently, nanoparticles have been reported to hold great potential for enhancing the efficiency of cancer treatment.

[0005] For example, Wang et al. demonstrated the potential to improve breast cancer treatment through cryosurgery by developing cryo-responsive polymer nanoparticles that release drugs upon cooling and generate localized heating under near-infrared (NIR) laser irradiation.

[0006] Kwak et al. reported that introducing thermally conductive inorganic nanoparticles into cryotherapy agents such as magnesium oxide, gold, silver, and iron oxide nanoparticles maximizes the degree of intracellular freezing, drug delivery, and imaging induction.

[0007] However, clinical application remains limited because intravenously injected nanoparticles have difficulty reaching the target tumor site, resulting in non-specific biodistribution and unsatisfactory therapeutic effects.

[0008] Therefore, research on the efficacy of low-temperature responsive nanomaterials and alternative delivery methods is necessary for the groundbreaking advancement of cryotherapy and optimal administration plans. The problem to be solved

[0010] The objective of the present invention is to provide a nanoplatform comprising upconversion nanoparticles.

[0011] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer comprising a nanoplatform that can be used for cryotherapy (Cryo) and photodynamic therapy (PDT) treatments.

[0013] The problems that the present invention aims to solve are not limited to the problem(s) mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below. means of solving the problem

[0015] To achieve the above objective, the present invention provides a nanoplatform comprising a core-shell structured upconversion nanoparticle comprising: a core made of NaYF4:Yb, Tm; and a shell made of NaYF4:Nd surrounding the core.

[0017] The above nanoplatform may further include a porous shell made of SiO2 surrounding the shell made of NaYF4:Nd.

[0018] The above porous shell comprises Chlorine e6, Zinc Phthalocyanine (ZnPC), Aminolevulinic Acid (ALA), Methyl Aminolevulinate, Temoporfin, Phthalocyanine, Protoporhap IX (PpIX), Allumera™, Cevira™, Hexvix™, Porfimer Sodium, Verteporfin, δ-Aminolevulinic Acid or 5-Aminolevulinic Acid, Temoporfin, Methyl Aminolevulinate, Hexaminolevulinate Hydrochloride, Talaporfin, Motexafin Lutetium, and 2-(1-Hexyloxyethyl)-2-Devinyl It may be loaded with at least one photosensitizer selected from the group consisting of pyropheophorbide-a (2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a), Photosens™, rostaporfin, BF-200 ALA, tetraphenyl chlorin disulfonate, and azadipyrromethene.

[0019] The above porous shell may have hyaluronic acid bound to it.

[0020] The size of the upconversion nanoparticles may be 10 to 100 nm.

[0021] The thickness of the porous shell may be 1 to 50 nm.

[0023] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising the above-mentioned nanoplatform.

[0024] The above cancer may be one or more selected from the group consisting of melanoma, lung cancer, head and neck cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, sarcoma, renal cell carcinoma, stomach cancer, esophageal cancer, anal canal cancer, cholangiocarcinoma, pancreatic cancer, liver cancer, cervical cancer, acute myeloid leukemia, chronic myeloid leukemia, skin cancer, lymphoma, thyroid cancer, bone marrow cancer, endometrial cancer, and brain tumor.

[0025] The above treatment may be used for a treatment selected from the group consisting of photodynamic therapy, cryotherapy, and combinations thereof.

[0027] In addition, the present invention provides a food composition for preventing or improving cancer, comprising the above-mentioned nanoplatform.

[0028] The above cancer may be one or more selected from the group consisting of melanoma, lung cancer, head and neck cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, sarcoma, renal cell carcinoma, stomach cancer, esophageal cancer, anal canal cancer, cholangiocarcinoma, pancreatic cancer, liver cancer, cervical cancer, acute myeloid leukemia, chronic myeloid leukemia, skin cancer, lymphoma, thyroid cancer, bone marrow cancer, endometrial cancer, and brain tumor. Effects of the invention

[0030] The nanoplatform comprising the upconversion nanoparticles of the present invention can be used in combination therapy with cryotherapy and photodynamic therapy (PDT) to treat skin melanoma more effectively, in particular.

[0032] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description of the invention or the claims. Brief explanation of the drawing

[0034] Figure 1 is a schematic diagram of a nanoplatform containing core-shell structure upconversion nanoparticles for the synergistic anticancer effect of transdermal Cryo-PDT treatment for cutaneous melanoma. Figure 2 schematically illustrates the optimization of reaction time for size uniformity of core(NaYF4:Yb,Tm)@shell(NaYF4:Nd) upconversion nanoparticles prepared in a single step through the Ostwald ripening method. Figure 3 is a low-resolution TEM image (scale bar: 1 μm) of UCNP@SiO2 nanoparticles to verify uniform coating and dispersion without severe aggregation. Figure 4 shows the results of the synthesis and characterization analysis of UCNPs and UCNP@SiO2, where a is a schematic diagram for the single-step synthesis of UCNPs containing NaYF4:Yb,Tm@NaYF4:Nd(core@shell), b is a TEM image (scale bar: 50 nm) of the synthesized UCNPs, and c is a SAED pattern (scale bar: 5 nm) of the UCNPs. -1 ) and d is the photoluminescence emission spectrum of UCNPs solution in cyclohexane after thawing and stored at room temperature (808 nm NIR laser irradiation, 1 W / cm² 2 ), the inserted image here is a digital image of the visually observable blue upconversion emission of a UCNP solution dissolved in cyclohexane at room temperature under 808 nm laser irradiation with the light on or off, e is a TEM image of UCNPs@SiO2 (scale bar: 50 nm), f is an FFT obtained from an HR-TEM image of UCNPs@SiO2, g is an X-ray diffraction pattern of the UCNPs@SiO2 sample and a reference pattern of hexagonal β-NaYF4 (JCPDS 16-0334), and h is a STEM-EDS elemental mapping result of the UCNPs@SiO2 sample (scale bar: 25 nm). Figure 5 shows the HAADF-STEM image and EDS elemental mapping image (scale bar: 25 nm) of UCNPs. Figure 6 is a schematic diagram of the upconversion process in the upconversion nanoparticles of the present invention. Figure 7 shows the particle size distribution of UCNPs and UCNPs@SiO2 samples (where σ g - Geometric standard deviation; d g,p - Geometric mean diameter, count number N = 300) and high-resolution TEM image of UCNPs@SiO2 (scale bar: 50 nm). Figure 8 is the photoluminescence emission profile of UCNPs before and after silica shell encapsulation under 808 nm laser excitation. FIG. 9 shows the results of confirming the characteristics of a nanoplatform prepared according to an embodiment of the present invention, wherein a is a schematic diagram for the preparation of the UCNP@SiO2-Ce6-HA nanoplatform, b is a TEM image of UCNPs@SiO2-Ce6 (scale bar: 50 nm), c is a TEM image of UCNPs@SiO2-Ce6-HA (scale bar: 50 nm), d is the FT-IR spectra of UCNPs@OA, UCNPs@SiO2-NH2, UCNPs@SiO2-Ce6, and UCNPs@SiO2-Ce6-HA, e is the normalized absorbance spectra of Ce6, UCNPs@SiO2, UCNPs@SiO2-Ce6, and UCNPs@SiO2-Ce6-HA, and f is under cryogenic conditions under 808 nm NIR laser irradiation using a DPBF sensor. Singlet oxygen generation profile of UCNPs@SiO2-Ce6-HA, g is the result of comparing the DPBF photodecomposition rate by UCNPs@SiO2-Ce6-HA under cryogenic (blue) and room temperature (red) conditions. Figure 10 is a Ce6 standard curve containing five data points, based on the absorption spectra of Ce6 solutions in dimethylformamide at various concentrations. Figure 11 shows the chemical structure of the HA-DAB conjugate and in deuterium oxide 1 This shows the H NMR spectrum. Here, m and n are each independently selected integers from 16 to 2500. Figure 12 shows the singlet oxygen generation profile results of the UCNPs@SiO2-Ce6-HA nanoplatform measured at room temperature under 808 nm laser irradiation using a DPBF sensor. Figure 13 shows the results of the relative cell viability (mean ± SD, n=3) of L929 and B16F10 cells quantitatively evaluated using various concentrations of UCNPs@SiO2-Ce6-HA nanoplatforms for 24 hours after culture. Figure 14 shows a the cell uptake rate results after injecting L929 and B16F10 cells with a UCNPs@SiO2-Ce6-HA nanoplatform at a concentration of 500 μg / mL for 4 hours after culture (scale bar: 150 μm), and b the quantitative fluorescence intensity results of a (n=3, **P < 0.01, ***P < 0.001 wrt UCNP@SiO2-Ce6-HA, and L929 vs. B16F10 cultured with UCNP@SiO2-Ce6-HA). Figure 15 shows the in vitro and in vitro characteristics of the nanoplatform, where a is the confocal microscopy result (blue: DAPI, red: Ce6, scale bar: 20 μm) of B16F10 cells treated with Ce6 and UCNPs@SiO2-Ce6-HA regardless of HA pre-incubation, and b is the result of various treatments: untreated (control), 808 nm laser only, UCNPs@SiO2-Ce6-HA only, and treatment with both 808 nm laser and UCNPs@SiO2-Ce6-HA (laser irradiation is 0.5 W / cm²). 2c shows the results of intracellular singlet oxygen detection in B16F10 cells after irradiation for 10 minutes at an intensity, where the green fluorescence of the DCF reflects the presence of singlet oxygen (scale bar: 150 μm); c shows the cell viability (mean ± SD, n=3, ***P < 0.001) of B16F10 cells cultured in various concentrations of UCNPs@SiO2-Ce6-HA (control group, 10, 50, 100, 200, 500 μg / mL) with or without 808 nm laser irradiation to confirm photodynamic therapy efficiency in vitro; and d shows various treatments: untreated (control group), 808 nm laser only, UCNPs@SiO2-Ce6-HA only, and treatment with both 808 nm laser and UCNPs@SiO2-Ce6-HA (laser irradiation was 0.5 W / cm²). 2 This is an image of a Live / dead analysis of B16F10 cells observed by a fluorescence microscope (green: calcein-AM, red: PI, scale bar: 300 μm) after irradiation for 10 minutes at an intensity, where green or red fluorescence indicates living cells or dead cells, respectively; e is a photograph observed via a confocal laser scanning microscope (scale bar: 50 μm) after skin tissues were collected by cryomicrotome 2, 4, 8, 12, and 24 hours after topically treating the porcine skin surface with UCNPs@SiO2-Ce6 and UCNPs@SiO2-Ce6-HA solutions (500 μg / mL) dispersed in PBS; and f is a photograph of skin tissues (SC: stratum corneum, Epi.:) collected from porcine skin treated with UCNPs@SiO2-Ce6-HA 2, 4, 8, 12, and 24 hours later. This is the result of observing the depth of penetration of the nanoplatform into the epidermis. Figure 16 shows images observed through a confocal laser scanning microscope after collecting skin tissues with a cryo-microtome at 2, 4, 8, 12, and 24 hours after topically treating porcine skin with a UCNPs@SiO2-Ce6 solution (500 μg / mL) dispersed in PBS (scale bar: 100 μm). Figure 17 shows a schematic diagram of a laser setup. Figure 18 shows the results of thermal imaging taken during a Cryo-PDT cycle including 1 minute Cryo followed by 1 minute Laser treatment. Each Cryo cycle includes 30 seconds of freeze-thaw cycles. Figure 19 shows the results of in vivo Cryo-PDT treatment, where a is the temperature change profile for the Cryo-PDT treatment cycle, with each cycle including 1 minute of Cryo and 1 minute of laser (total 10 cycles), and according to the FLIR infrared camera user manual, the lowest temperature measurement range is limited to -20 ℃, and b is 500 μg / mL of UCNPs@SiO2-Ce6 and UCNPs@SiO2-Ce6-HA solutions dispersed in PBS. Images observed using a confocal laser scanning microscope after frozen sections of melanoma tumor tissue were topically treated in vivo at a concentration of (scale bar: 150 μm); dotted lines indicate skin / tumor boundaries; c is a representative photograph of melanoma tumor tissue collected from C57BL / 6 mice after various treatments: (1) control; (2) UCNPs@SiO2-Ce6-HA; (3) cryo; (4) laser; (5) cryo + UCNPs@SiO2-Ce6-HA; (6) laser + UCNPs@SiO2-Ce6-HA; and (7) Cryo + Laser + UCNPs@SiO2-Ce6-HA, d is the relative change in body weight and e is the relative tumor growth curve after various treatments for 14 days, error bars represent the standard deviation (SD) of 4 (n=4) individual sample measurements, statistical significance was evaluated using ANOVA (**P < 0.01 and ***P < 0.001), and f is the result of H&E and TUNEL staining of tumor tissue after various treatments (scale bar: 150 μm). Specific details for implementing the invention

[0035] The terms used in this specification have been selected based on currently widely used general terms whenever possible, taking into account their functions in the present invention; however, these terms may vary depending on the intent of those skilled in the art, case law, or the emergence of new technologies. Additionally, in specific cases, terms have been arbitrarily selected by the applicant, and in such cases, their meanings will be described in detail in the relevant description of the invention. Therefore, terms used in this specification should be defined not merely by their names, but based on their meanings and the overall content of the present invention.

[0036] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which this invention pertains. Terms generally understood should be interpreted as having the same meaning as they have in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this invention.

[0037] Numerical ranges include the numerical values ​​defined in the present invention. All maximum numerical limits given throughout this specification include all lower numerical limits as clearly written. All minimum numerical limits given throughout this specification include all higher numerical limits as clearly written. All numerical limits given throughout this specification will include all better numerical ranges within a wider numerical range, as clearly written.

[0039] The present invention will be described in detail below.

[0040] In one embodiment of the present invention, a nanoplatform comprising upconversion nanoparticles of a core-shell structure is provided.

[0041] In one embodiment, a schematic diagram of a nanoplatform comprising core-shell structured upconversion nanoparticles for combination therapy of cryotherapy and photodynamic therapy (or referred to as “Cryo-PDT therapy”) is shown in FIG. 1. Specifically, FIG. 1 is a schematic diagram of a nanoplatform comprising core-shell structured upconversion nanoparticles for the synergistic anticancer effect of transdermal Cryo-PDT therapy against cutaneous melanoma.

[0042] In one embodiment, the upconversion nanoparticle of the core-shell structure (or referred to as “UCNP” or “UCNPs”) may comprise a core composed of NaYF4:Yb, Tm; and a shell composed of NaYF4:Nd surrounding the core. Herein, the core and the shell have the chemical formula M containing lanthanide ions. 1 M 2 It can be formed from a composition consisting of a rare earth metal fluoride according to F4. In the above chemical formula, M 1 It may include one or more elements selected from the group consisting of lithium (Li), sodium (Na), potassium (K), (Rb), cesium (Cs), francium (Fr), beryllium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), and barium (Ba), and M 2 It is a rare earth element and may contain one or more elements selected from the group consisting of scamdium (Sc), yttrium (Y), lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), diprosium (Dy), holmium (Ho) and erbium (Er), and the presence of the rare earth element in the above chemical formula may be included in an amount of 0 mol% to 99 mol%.

[0043] In one embodiment, the UCNP can improve upconversion luminescence efficiency due to an increase in near-infrared absorption at 808 nm.

[0044] In one embodiment, the nanoplatform may further include a porous shell made of SiO2 surrounding the shell made of NaYF4:Nd. The porous shell made of SiO2 may be formed on the UCNP to stabilize the UCNP in a biological environment and to conjugate with the PDT photosensitizer described later.

[0045] In one embodiment, the porous shell may be loaded with a PDT photosensitizer, for example, Chlorine e6, zinc phthalocyanine (ZnPC), aminolevulinic acid (ALA), methyl aminolevulinate, temoporfin, phthalocyanine, protoporhap IX (PpIX), Allumera™, Cevira™, Hexvix™, porfimer sodium (Photofrin™), verteporfin (Visudyne™), δ-aminolevulinic acid or 5-aminolevulinic acid (Levulan™), temoporfin (Foscan™), methyl aminolevulinate (Metvix™), hexaminolevulinate hydrochloride hydrochloride (Cysview™)), talaporfin (Laserphyrin™)), motexafin lutetium (Antrin™)), 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (Photochlor™)), Photosens™ (mixture of sulfonated aluminum phthalocyanines with various degrees of sulfonation), rostaporfin (Photrex™, SnET2, Purlytin™)), BF-200 ALA (nanoemulsion BF-200 with 5-aminolevulinic acid), tetraphenylchlorin disulfonate (TPCS2a,At least one photosensitive agent selected from the group consisting of Amphinex™)) and azadipyrromethene may be loaded, and upon irradiation with a red laser, singlet oxygen (, 1 O2) can be generated. Cancer cell viability is reduced by the singlet oxygen mentioned above. A suitable photosensitizer can be selected according to the purpose of the invention.

[0046] In one embodiment, the porous shell may be bound to hyaluronic acid. The binding of hyaluronic acid to the porous shell enables the nano-platform to be transdermally delivered into cancer tissue and further stabilized in a physiological environment.

[0047] The above hyaluronic acid can be conjugated or bonded to a porous shell using EDC-NHS coupling. In one embodiment, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) refers to a coupling reaction used for immobilization, etc., by binding with N-hydroxysuccinimide (NHS) or sulfo-NHS.

[0048] The size of the upconversion nanoparticles may be 10 to 100 nm, specifically 20 to 40 nm, and more specifically 30 to 35 nm.

[0049] The thickness of the porous shell may be 1 to 50 nm.

[0051] In one embodiment of the present invention, a pharmaceutical composition for cancer prevention or treatment comprising the nanoplatform is provided.

[0052] The above-mentioned nanoplatform is identical to the nanoplatform described above, and any content that overlaps with that described in the nanoplatform described above will not be explained again.

[0053] As used herein, the term "photodynamic therapy or photodynamic therapy (PDT)" refers to a process in which a photosensitizer, after being activated by light, undergoes a chemical reaction with molecular oxygen to produce singlet oxygen, 1 It refers to a next-generation therapeutic method that generates O2, and this singlet oxygen selectively destroys target cells or tissues.

[0054] As used herein, the term “cryotherapy” refers to a treatment method that selectively destroys or treats abnormal skin tissue or induces a change in the local immune response by rapidly freezing skin lesions using a medium at very low temperatures.

[0055] As used herein, the term “cancer” refers collectively to diseases caused by cells having aggressive characteristics in which cells divide and grow disregarding normal growth limits, invasive characteristics in which they infiltrate surrounding tissues, and metastatic characteristics in which they spread to other parts of the body. The cancer may be one or more selected from the group consisting of, for example, melanoma, lung cancer, head and neck cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, sarcoma, renal cell carcinoma, gastric cancer, esophageal cancer, anal canal cancer, cholangiocarcinoma, pancreatic cancer, liver cancer, cervical cancer, acute myeloid leukemia, chronic myeloid leukemia, skin cancer, lymphoma, thyroid cancer, bone marrow cancer, endometrial cancer, and brain tumor; according to one embodiment or experimental example of the present invention, it may be melanoma, but is not limited thereto.

[0056] The above treatment may be used for a treatment selected from the group consisting of photodynamic therapy, cryotherapy, and combinations thereof.

[0058] In one embodiment, the pharmaceutical composition may further comprise a suitable carrier, excipient, and diluent commonly used in the manufacture of pharmaceutical compositions. The excipient may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, humectants, film-coating materials, and controlled-release additives.

[0059] In one embodiment, the pharmaceutical composition may be formulated and used in the form of a powder, granule, sustained-release granule, enteric granule, liquid, eye drops, ellipsis, emulsion, suspension, ethanol, troche, fragrance, limonene admixture, tablet, sustained-release tablet, enteric tablet, sublingual tablet, hard capsule, soft capsule, sustained-release capsule, enteric capsule, pill, tincture, soft extract, dry extract, fluid extract, injection, capsule, irrigation solution, warning agent, lotion, paste, spray, inhalant, patch, sterile injectable solution, or aerosol, etc., according to a conventional method, and the external agent may have a formulation such as a cream, gel, patch, spray, ointment, warning agent, lotion, liniment, paste, or cataplasma.

[0060] In one embodiment, the product may be formulated and used in the form of a powder, granule, sustained-release granule, enteric granule, liquid, eye drops, ellipsis, emulsion, suspension, ethanol, troche, fragrance, limonene admixture, tablet, sustained-release tablet, enteric tablet, sublingual tablet, hard capsule, soft capsule, sustained-release capsule, enteric capsule, pill, tincture, soft extract, dry extract, fluid extract, injection, capsule, irrigation solution, warning agent, lotion, paste, spray, inhalant, patch, sterile injectable solution, or aerosol, etc., according to each of the above conventional methods, and the product may have a formulation such as a cream, gel, patch, spray, ointment, warning agent, lotion, liniment, paste, or cataplasma.

[0061] In one embodiment, carriers, excipients, and diluents that may be included in the pharmaceutical composition may include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0062] When formulating, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0063] Excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium monohydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, refined lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethylcellulose (HPMC), HPMC 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, primogel, etc., as additives to the above tablets, powders, granules, capsules, pills, and lozenges; Gelatin, gum arabic, ethanol, agar powder, cellulose phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium casein, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch paste, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc., may be used as binders, and hydroxypropylmethylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Calcium carboxymethylcellulose, calcium citrate, sodium lauryl sulfate, anhydrous silica, 1-hydroxypropylcellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, D-sorbitol solution, hard anhydrous silica, etc. disintegrants;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium pods, kaolin, petroleum jelly, sodium stearate, cocoa paste, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silica, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and hard anhydrous silica may be used.

[0064] In one embodiment, as additives to the liquid formulation, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearic acid sucrose, polyoxyethylene sorbitol fatty acid esters (tween esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, water ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc. may be used.

[0065] The above syrup may use a solution of white sugar, other sugars or sweeteners, etc., and, if necessary, flavorings, coloring agents, preservatives, stabilizers, suspending agents, emulsifiers, viscosity enhancers, etc.

[0066] Purified water may be used in the above emulsion, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0067] The above suspension agent may use suspending agents such as acacia, tragacanthus, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, HPMC 2910, etc., and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

[0068] In one embodiment, the injectable agent comprises solvents such as distilled water for injection, 0.9% sodium chloride injection solution, Ringer's injection solution, dextrose injection solution, dextrose + sodium chloride injection solution, PEG, lactated Ringer's injection solution, ethanol, propylene glycol, non-volatile oils—sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; solubilizing agents such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nijungtinamide, hexamine, and dimethylacetamide; and buffers such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums. It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; sulfating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid, and sodium bisulfite acetone; non-inflammatory agents such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium CMC, sodium alginate, Tween 80, and aluminum monostearate.

[0069] The above suppositories contain cocoa dough, lanolin, Witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, lanette wax, glycerol monostearate, Tween or Spandex, Imhausen, Monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydrokote SP, S-70-XXA, S-70-XX75 (S-70-XX95), and Hydrokote. Bases such as 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Masa-MF, Masupol, Masupol-15, Neosupostal-N, Paramount-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), suppository base type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wekovi (W, R, S, M, Fs), and Tegestor triglyceride base (TG-95, MA, 57) may be used.

[0070] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the extract. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used.

[0071] Liquid preparations for oral administration include suspensions, liquid formulations, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as humectants, sweeteners, flavorings, and preservatives, may be included.

[0072] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used.

[0073] In one embodiment, the pharmaceutical composition is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined according to factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0074] In one embodiment, the pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects, taking all of the above factors into consideration, and this can be easily determined by a person skilled in the art to which the present invention pertains.

[0075] In one embodiment, the pharmaceutical composition may be administered to an individual by various routes. All modes of administration are expected, for example, oral administration, subcutaneous injection, intraperitoneal administration, intramuscular injection, intrathecal (intradural) injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, ear administration, nasal administration, inhalation, spray through the mouth or nose, skin administration, transdermal administration, etc.

[0076] In one embodiment, the pharmaceutical composition is determined according to the type of active ingredient drug, along with various relevant factors such as the disease to be treated, the route of administration, the patient's age, gender, weight, and the severity of the disease.

[0078] In one embodiment of the present invention, a food composition for preventing or improving cancer is provided, comprising the nanoplatform.

[0079] The above-mentioned nanoplatform is identical to the nanoplatform described above, and any content that overlaps with that described in the nanoplatform described above will not be explained again.

[0080] The above cancer may be one or more selected from the group consisting of melanoma, lung cancer, head and neck cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, sarcoma, renal cell carcinoma, stomach cancer, esophageal cancer, anal canal cancer, cholangiocarcinoma, pancreatic cancer, liver cancer, cervical cancer, acute myeloid leukemia, chronic myeloid leukemia, skin cancer, lymphoma, thyroid cancer, bone marrow cancer, endometrial cancer, and brain tumor.

[0081] In one embodiment, when the nanoplatform is used as a food additive, the nanoplatform may be added as is or used together with other food or food ingredients, and may be used appropriately according to conventional methods. The amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing food or beverages, the nanoplatform of the present invention may be added in an amount of 15% by weight or less, or 10% by weight or less, relative to the raw material. However, in the case of long-term consumption for the purpose of health and hygiene or health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount greater than the above range.

[0082] There are no specific restrictions on the types of the above-mentioned foods. Examples of foods to which the above-mentioned substance may be added include meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and include all health functional foods in the conventional sense.

[0083] In one embodiment, the health drink composition may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. The natural carbohydrates mentioned above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As sweeteners, natural sweeteners such as taumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used. The proportion of the natural carbohydrates is generally about 0.01-0.20g or about 0.04-0.10g per 100 mL of the composition of the present invention.

[0084] In addition to the above, the composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Furthermore, the composition of the present invention may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. These ingredients may be used independently or in combination. Although the proportion of these additives is not critical, it is generally selected in the range of 0.01 to 0.20 parts by weight per 100 parts by weight of the composition of the present invention.

[0085] In one embodiment, “individual” means a subject requiring treatment for a disease, and more specifically, means mammals such as human or non-human primates, mice, rats, dogs, cats, horses, and cattle.

[0086] In one embodiment, “administration” means providing a predetermined composition of the present invention to an individual by any appropriate method.

[0087] In one embodiment, “prevention” refers to any act of suppressing or delaying the onset of a target disease, “treatment” refers to any act of improving or beneficially altering the target disease and the associated metabolic abnormality symptoms by administering a pharmaceutical composition according to the present invention, and “improvement” refers to any act of reducing parameters related to the target disease, such as the severity of symptoms, by administering a composition according to the present invention.

[0089] The above description explains the technical concept of the present invention using one embodiment, and those skilled in the art to which the present invention pertains will be able to make various modifications and variations within the scope of the essential characteristics of the present invention. Accordingly, the embodiments described in this invention are intended to explain, not limit, the technical concept of the present invention, and the scope of the technical concept of the present invention is not limited by such embodiments. The scope of protection of the present invention shall be interpreted by the claims, and all technical concepts within an equivalent scope shall be interpreted as being included within the scope of rights of the present invention.

[0090] <Example>

[0091] ingredient

[0092] Reagents and solvents were purchased from commercial suppliers and used without purification. Yttrium(III) acetate tetrahydrate (Y(CH3COO) 3 · 4H2O, 99.9%, ytterbium(III) acetate hydrate (Yb(CH3COO) 3 · nH2O, 99.9%, thulium(III) acetate hydrate (Tm(CH3COO)3 · nH2O, 99.9%) and neodymium(III) acetate hydrate (Nd(CH3COO)3 · nH2O (99.9%), oleic acid (OA, tech. 90%), 1-octadecene (ODE, tech. 90%), ammonium fluoride (> 98%), cyclohexane (ACS grade), tetraethoxysilane (TEOS, 99.9%) and 1,3-diphenylisobenzofuran (97%) were all purchased from Alfa Aesar.

[0093] Methanol (99.9%), absolute ethanol (99.9%), sodium hydroxide pellets (>97%), and sodium chloride were purchased from Daejeong Chemical. Ammonium nitrate (≥98%), cetyltrimethylammonium bromide (CTAB, ≥98%), (3-aminopropyl)triethoxysilane (APTES, 99%), and 4',6-diamidino-2-phenylindole (DAPI) were purchased from Sigma Aldrich.

[0094] Sodium hyaluronate (HA, MW=100 kDa) was purchased from SNvia Co., Ltd. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, > 98%), N-hydroxysuccinimide (NHS, 98%), and diaminobutane (DAB) were purchased from TCI (Tokyo Chemical Industries Co., Ltd.). Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), penicillin, and phosphate-buffered saline (PBS) were purchased from Invitrogen Co., Ltd. Chlorine e6 was obtained from MedChem Express LLC. 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) was purchased from Cayman Chemical Company.

[0095] Cell counting kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies. Max-View™ Live / Dead Staining Kit (Calcein-AM / PI) was purchased from Biomax Co., Ltd. Mouse fibroblast (L929) and melanoma (B16F10) cell lines were purchased from the Korea Cell Line Bank.

[0097] Synthesis of upconversion nanoparticles (referred to as "UCNPs")

[0098] UCNPs were synthesized through the Ostwald aging method using the high-temperature co-precipitation method.

[0099] Specifically, a lanthanide precursor solution-core solution (hereinafter referred to as “Solution A”): Y(CH3COO)3 (0.2 M, 4 mL), Yb(CH3COO)3 (0.2 M, 1.9 mL), and Tm(CH3COO)3 (0.002 M, 0.5 mL); and a shell solution (hereinafter referred to as “Solution B”): Y(CH3COO)3 (0.2 M, 4 mL), and Nd(CH3COO)3 (0.2 M, 1.0 mL) were each prepared in a 250 mL three-necked round-bottom flask. The round-bottom flask was heated to 110°C for 10 minutes to evaporate the water, and then a mixture of OA (6.0 mL) and ODE (15.0 mL) was added to Solution A and Solution B, respectively.

[0100] Then, the flask containing solution A was connected to a Schlenk line for degassing and nitrogen purging. The flask containing solution A was maintained at 150°C for 30 minutes under a weak nitrogen flow to completely dissolve the lanthanide salt in the mixture of OA and ODE. Next, 0.148 g of NH4F and 0.1 g of NaOH were dissolved in 9 mL of methanol and injected into the flask containing solution A. The flask was then heated to 110°C to evaporate the methanol, followed by degassing for 20 minutes. After the methanol was completely removed, the flask containing solution A was heated at 300°C for 1 hour under a nitrogen (N2) atmosphere. Subsequently, the flask containing solution B was precipitated, degassed, and heated to 150°C (in the presence of N2), then injected into the flask containing solution A at 300°C, and the reaction was carried out for 1, 1.5, and 2 hours for size focusing (Fig. 2, where particle size distribution count N=300 and the scale bar in the TEM image represents 100 nm). Overpressure in the reactor system was prevented using a double nitrogen balloon system. After cooling to room temperature, acetone was added to precipitate the UCNP, which was then extracted by centrifugation. After washing several times with acetone, the solution was finally redispersed in cyclohexane (20 mL) for further use.

[0102] UCNP coated with mesoporous silica("UCNP@SiO 2 ” or "UCNPs@SiO 2 Compound of (referred to as )

[0103] First, 4 mL of a UCNP solution dispersed in cyclohexane was added to 40 mL of DI water containing 0.32 g of CTAB under ultrasonic treatment (Sonics VCX-750 Vibra-Cell Ultrasonic Liquid Processor, Sonics & Materials, Inc., Newton, CT, USA). The cyclohexane was further removed by gently heating in a bath-ultrasonic device at 50°C for 2 hours. Subsequently, 20 mL of DI water was added to the dispersion, the pH was adjusted to 10.0 using a 0.1 M NaOH solution, and the mixture was further homogenized by ultrasonic treatment for 30 minutes. Next, TEOS and anhydrous ethanol were thoroughly mixed in a volume ratio of 1:4, respectively, and then added dropwise to the solution while continuously applying ultrasound to prevent aggregation (Fig. 3).

[0104] Then, the solution was maintained at 30°C for 24 hours while vigorously stirring. The synthesized UCNP@SiO2 was obtained by centrifugation at 8000 rpm for 20 minutes and washed several times with ethanol. The CTAB surfactant was removed via ion exchange. The UCNP@SiO2 was transferred into a flask containing 0.3 g NH4NO3 dissolved in 40 mL of ethanol. The flask was placed under ultrasound at 50°C for 1 hour. The CTAB extraction process was repeated twice. Next, the obtained UCNPs@SiO2 was grafted with amine groups to further load the therapeutic component.

[0105] First, UCNP@SiO2 was redispersed in an ethanol / water (95:5 by weight) solvent mixture, 100 μL of APTES was added, and the mixture was vigorously stirred under reflux at 70 °C for 2 hours. Finally, UCNP@SiO2-NH2 was separated by centrifugation at 8000 rpm for 20 minutes and redispersed in 4 mL of DI water for further use.

[0106] For bioimaging purposes, UCNP@SiO2-NH2 nanoparticles were labeled with a FITC photosensitive agent by simple stirring overnight. Labeling variation was demonstrated by a change in sample color from white to yellow.

[0108] Loading Ce6 photosensitizer onto UCNPs coated with mesoporous silica("UCNP@SiO 2 -Ce6” or "UCNPs@SiO 2 -Referred to as “Ce6”)

[0109] 3 mg of Ce6 was dispersed in 4 mL of DI water, and its carboxyl group was activated by the EDC / NHS reaction. Then, 4 mL of UCNPs@SiO2-NH2 (10 mg) dispersed in DI water was added to a solution containing Ce6 and sonicated for 10 minutes to form a homogeneous dispersion.

[0110] Then, to avoid interference from ambient light, the dispersion was placed in an amber glass vial and vigorously stirred at room temperature. After a 24-hour reaction, UCNPs@SiO2-Ce6 was precipitated by centrifugation at 8000 rpm for 20 minutes. After removing excess Ce6 by washing three times with DI water, the unreacted Ce6 was quantified by measuring the absorption spectrum of the collected supernatant.

[0111] The loading capacity was calculated based on the absorbance correction plot of a solution with a known Ce6 concentration. The loading capacity was confirmed to be 29.2 wt% based on the calibration curve in Fig. 10. Finally, the generated UCNPs@SiO2-Ce6 was freeze-dried and stored in a dark room at -20°C.

[0113] HA-DAB synthesis

[0114] To synthesize HA-DAB, HA with a MW of 100 kDa was dissolved in an ethanol / water (1:1 volume ratio) solvent mixture, and then DAB (40 molar ratio of HA repeating units) was dissolved in the HA-containing solution.

[0115] Next, EDC and NHS (2 molar ratio of HA) were introduced into the formed HA-containing solution to activate the carboxyl groups of HA. The pH of the mixed solution was adjusted to 5.5 using a 1N aqueous HCl solution. The reaction was carried out at room temperature for 24 hours with vigorous stirring, and the resulting HA-DAB conjugate was dialyzed against a 100 mM NaCl solution for 3 days, a 25% ethanol solution for 1 day, and DI water for 1 day.

[0116] The purified conjugate solution was freeze-dried for 3 days. The effective degree of HA modification is shown in the proton nuclear magnetic resonance (Fig. 11 (AVANCE NEO 500, Bruker, Germany)). 1 It was analyzed using the H NMR spectrum.

[0118] UCNPs@SiO 2 -Ce6-HA("UCNP@SiO 2 -Ce6-HA” or "UCNPs@SiO 2 Fabrication of a nano-platform of (referred to as “-Ce6-HA”)

[0119] The prepared HA-DAB was conjugated to the surface of UCNPs@SiO2-Ce6 (10 mg) via an EDC / NHS reaction. The mixture was prepared in 10 mL of DI water and stirred in an amber glass vial at room temperature for 24 hours.

[0120] The resulting suspension was dialyzed against DI water for 3 days in a dialysis bag with a molecular weight cutoff of 7 kDa to remove unreacted molecules. The final freeze-dried product was stored in a dark place at -20°C for future use.

[0122] <Experimental Example>

[0123] Characteristic evaluation

[0124] The sample morphology image was confirmed using an 80 kV transmission electron microscope (TEM, Talos, FEI, Lausanne, Switzerland).

[0125] High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM), energy dispersive spectroscopy (EDS) mapping, and selected area electron diffraction (SAED) were performed using 200 kV TEMs such as JEM-2100F and JEM-ARM200F (JEOL, Tokyo, Japan).

[0126] Carbon contamination of the TEM grid was removed using an ion cleaner JIC-410 at 285 V for 5 minutes (JEOL, Tokyo, Japan).

[0127] Crystallographic analysis was performed using Vesta software (Vesta Software Group, Wallingford, UK).

[0128] The upconversion emission spectrum was measured using a spectrofluorometer (FL-1039, HORIBA Scientific Co., Kyoto, Japan) with an 808 nm infrared diode laser system as the excitation source. The laser power density was adjusted using a power meter (843-R, Newport Corp., Irvine, USA).

[0129] Qualitative analysis of functional groups on the surface of nanoparticles was performed using a Spectrum Two Fourier Transform Infrared Spectrometer (Perkin Elmer Inc., Waltham, MA, USA).

[0130] The crystal structure of UCNPs@SiO2powder was analyzed using a PANalytical X'Pert Pro X-ray diffractometer.

[0131] UV / vis absorption spectra were analyzed using a Scinco Mega-800 spectrophotometer (Seoul, Korea). The lanthanide composition of UCNP was analyzed using an inductively coupled plasma triple quadrupole mass spectrometer (iCap-TQ model, Thermo Fisher Scientific, Waltham, MA, USA). Dynamic light scattering and zeta potential measurements were performed using a Malvern Pananalytical Zetasizer Nano ZS90 instrument (Malvern, UK).

[0132] Particle size distribution analysis was performed using ImageJ software (Rasband, WS, ImageJ, US National Institutes of Health, Bethesda, Maryland, USA).

[0133] Geometric mean diameter d g,p and geometric standard deviation σ g It was calculated through the equation shown below:

[0134] (1)

[0135] (2)

[0137] Here, N is the total number of samples and d i is the particle size measured in the TEM image.

[0139] Extracellular detection of reactive oxygen species

[0140] 1 mL of aqueous UCNPs@SiO2-Ce6-HA (1 mg / mL) solution was mixed with 1 mL of DPBF solution dissolved in ethanol in a 4 mL cuvette, and then the sample was subjected to 0.5 W / cm² 2 It was exposed to an 808 nm laser at an output density.

[0141] UV-Vis absorption was measured at approximately 410 nm for 10 minutes (0, 2, 4, 6, 8, 10 minutes). The low-temperature effect was confirmed by maintaining the cuvette at -80°C for 1 minute prior to laser irradiation.

[0143] Cell absorption experiment

[0144] Cellular uptake was evaluated using L929 and B16F10 cell lines purchased from the Korean Cell Line Bank (Seoul, Korea). 1 × 10⁶ cells were used per well. 5 The cells were cultured in a 12-well plate at a cell density. The cells were cultured for one day at 37°C in a standard medium containing 10% FBS and 1% antibiotics in an environment containing 5% CO2.

[0145] Then, the cells were cultured with a nanoparticle suspension at a concentration of 500 μg / mL for 4 hours. Before fixation with 4% paraformaldehyde, the cells were washed twice with PBS solution.

[0146] Next, cells were stained with DAPI for 10 minutes before imaging with a confocal laser scanning microscope (ZEISS LSM 800, Oberkochen, Germany). To understand the roles of HA receptors CD44 and LYVE-1 during the influx of nanoparticles into cells, competitive uptake inhibition was performed in B16F10 cell lines by adding free HA (100 kDa, 10 mg / mL, 200 μL) for 30 minutes.

[0148] Live / Dead Cell Evaluation

[0149] 0.5 × 10⁶ B16F10 cells per well in a 24-well plate 5 The cells were cultured at a density of dog cells. After culturing the cells for 24 hours, they were cultured with a nanoparticle suspension at a concentration of 500 μg / mL for 4 hours, and then subjected to 0.5 W / cm² with an 808 nm laser. 2 The cells were irradiated for 10 minutes at an output density. Before imaging with a fluorescence microscope (EVOS M5000, Thermo Fisher Scientific, Waltham, MA, USA), the cells were cultured with Live / Dead cell analysis solution for 10 minutes.

[0151] Cell viability analysis by CCK analysis

[0152] The cytotoxicity of the samples was evaluated by the CCK-8 (tetrazolium salt) assay. 1 × 10⁶ B16F10 cells were used per well. 4 The cells were cultured in 96-well plates at a density of dog cells. The cells were cultured for one day at 37°C in a standard medium containing 10% FBS and 1% antibiotics in an environment containing 5% CO2. Then, the cells were cultured for 24 hours with nanoparticle suspensions at different concentrations (10, 50, 100, 200, and 500 μg / mL).

[0153] Next, the cell medium was replaced, and 10 μL of the freshly prepared CCK-8 assay was added to each well. After incubation for 4 hours, the absorbance of CCK was measured at 450 nm using a microplate reader. Cell viability quantification was performed according to the manufacturer's instructions.

[0155] Evaluation of intracellular singlet oxygen production

[0156] 0.5 × 10⁶ B16F10 cells per well in a 24-well plate 5 The cells were cultured at a density of dog cells. After culturing the cells for 24 hours, they were cultured with a nanoparticle suspension at a concentration of 500 μg / mL for 4 hours, and then subjected to 0.5 W / cm² with an 808 nm laser. 2 The cells were irradiated for 10 minutes at an output density. Before imaging with a fluorescence microscope, the cells were cultured with the DCFH-DA probe for 30 minutes.

[0158] In vitro photodynamic therapy

[0159] 1 × 10⁶ B16F10 cells per well in a 96-well plate 4 Canine cells were cultured at a cell density for 24 hours. Then, the cells were cultured with nanoparticle suspensions at various concentrations (10, 50, 100, 200, and 500 μg / mL). After 4 hours of incubation, 0.5 W / cm² except for the control group 2The cells were irradiated with an 808 nm laser for 10 minutes at an output density. After laser irradiation, the cells were incubated for 12 hours, then washed three times with PBS and incubated with a CCK-8 assay to quantify relative cell viability.

[0161] UCNPs@SiO 2 - Transdermal delivery of Ce6-HA nanoplatform

[0162] The penetration ability of the UCNPs@SiO2-Ce6-HA nanoplatform across the skin layer was evaluated using freshly cut pig skin obtained from a local slaughterhouse. The UCNPs@SiO2-Ce6 and UCNPs@SiO2-Ce6-HA nanoplatforms were dispersed in PBS at a concentration of 500 μg / mL and then topically applied to the surface of pig skin cut into 0.5 x 0.5 cm pieces using a cryo-microtome (Leica, CM1860, Wetzlar, Germany).

[0163] Transdermal delivery was evaluated after 2, 4, 6, 12, and 24 hours using a confocal laser scanning microscope (ZEISS LSM 800, Oberkochen, Germany). Fluorescence intensity was measured using ImageJ software.

[0165] In vivo antitumor effects of cryo-photodynamic (Cryo-PDT) therapy

[0166] Female C57BL / 6NCrlOri mice aged 5–6 weeks were purchased from Orient Bio Co., Ltd. (Seongnam, Korea). All mice were maintained under pathogen-free conditions, and all procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Pusan ​​National University. Before treatment, mice were randomly divided into several groups.

[0167] To evaluate the antitumor Cryo-PDT effect, 5 × 10 mice 6 A tumor was induced by subcutaneously injecting B16F10 melanoma cells at a density, and B16F10 cells were suspended in DMEM and injected into the right dorsal flank.

[0168] One week after inoculating tumor cells, mice were randomly divided into seven treatment groups (n = 4 per group): Group 1: PBS (Control), Group 2: UCNPs@SiO2-Ce6-HA, Group 3: Cryo, Group 4: Laser, Group 5: Cryo + UCNPs@SiO2-Ce6-HA, Group 6: Laser + UCNPs@SiO2-Ce6-HA (PDT), Group 7: Cryo + Laser + UCNPs@SiO2-Ce6-HA (Cryo-PDT).

[0169] During the treatment phase, the average tumor volume is 70 mm 3 It was. Tumor growth was monitored using the following formula: Tumor Volume = A × B 2 / 2, where A and B are the maximum and minimum diameters of the tumor, respectively. A solution of UCNPs@SiO2-Ce6-HA (500 μg / mL, 50 μL) in PBS was topically administered to the tumor surfaces of groups 2, 5, 6, and 7 for 30 minutes.

[0170] Cryotherapy for groups 3 and 5 was performed using sterile cotton pads pre-soaked in liquid nitrogen, which were then applied to the tumor site for 10 minutes. For laser groups 4 and 6, an 808 nm laser was applied at 0.5 W / cm² 2 Irradiation was performed for 10 minutes at a power density. In particular, the Cryo-PDT combination therapy in Group 7 was performed over 10 consecutive cycles, with each cycle consisting of 1 minute of Cryo (liquid nitrogen) therapy and 1 minute of laser therapy (0.5 W / cm²). 2 It was composed of ). Each cryo cycle includes 30-second freeze-thaw cycles.

[0171] The laser setup is illustrated in Fig. 17. The laser power density was adjusted using a power meter (843-R, Newport, USA). Thermal images were captured using a FLIR E6390 infrared camera (Teledyne FLIR LLC, Wilsonville, USA). Tumor size and mouse body weight were measured and recorded over 14 days after treatment. Relative tumor volume was calculated as the ratio of the tumor volume to the initial tumor volume.

[0173] Histological analysis of tumor cell death

[0174] After 14 days of treatment, tumor tissues were collected from all treatment groups, washed with PBS, and stored in 4% paraformaldehyde. The tumor tissues were incised and embedded in paraffin. To assess the degree of tumor apoptosis, a hematoxylin and eosin staining kit (H&E, Tissue Protech.) and a terminal-deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay kit (DeadEnd™ Fluorometric TUNEL System, Promega) were used. H&E and TUNEL staining procedures were performed according to the manufacturer's protocols.

[0176] Statistical analysis

[0177] Statistical analysis was performed using one-way ANOVA with GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA), and values ​​of *, P < 0.05; **, P < 0.01; and ***, P < 0.001 were considered significant. All data were expressed as mean ± standard deviation (SD) obtained from at least three independent experiments.

[0179] Confirmation of the characteristics of upconversion nanoparticles

[0180] Figure 4a shows a schematic diagram of the synthesis pathway of the upconversion nanoparticles of the present invention. The upconversion nanoparticles of the present invention can have their sodium fluoride content precisely controlled during synthesis by injecting solution B, which is a pre-forming precipitating agent, into solution A.

[0181] In particular, since a pressure increase after injection due to residual methanol and moisture is inevitable, a dual nitrogen (N2) gas balloon system was applied during synthesis to stabilize the pressure. In addition, the effect of synthesis time after injection on particle size distribution was investigated, and the results are shown in Figure 2.

[0182] Referring to Fig. 2, it can be seen that synthesis for 2.5 hours yields an optimal uniform size, which is accompanied by a sharp size distribution profile and a geometric standard deviation (σ) of 1.046. g It was confirmed as ).

[0183] Figure 4b is a TEM image of the upconversion nanoparticles of the present invention, and with reference to Figure 4b, it can be seen that monodisperse upconversion nanoparticles with a particle size of ~32 nm were prepared.

[0184] As a result, the above synthesis conditions showed that the final homogeneous core@shell (NaYF4:Yb,Tm@NaYF4:Nd) nanoparticles were formed.

[0185] To verify the formation of a hexagonal crystal structure, the selected region electron diffraction (SAED) pattern of a single NaYF4:Yb,Tm@NaYF4:Nd nanoparticle was analyzed using high-resolution transmission electron microscopy (HR-TEM).

[0186] As shown in FIG. 4c, the interplanar spacing within the ring pattern was measured to be 5.185 Å, 2.999 Å, 1.960 Å, and 1.740 Å, which corresponded well with the planes (100), (110), (210) and (002) of the hexagonal β-NaYF4.

[0187] In addition, the presence of Y, Yb, Tm, and Nd elements was confirmed by mapping the elemental distribution using HAADF-EDS (high-angle annular dark-field imaging coupled with energy dispersive spectroscopy), and the results are shown in Figure 5.

[0188] According to ICP-OES (Inductively Coupled Plasma Optical Emission Spectrometry) analysis, the molar ratio of Y:Yb:Tm:Nd in UCNP was 81.5:9.0:0.1:9.4 (Table 1). All these results confirmed the successful synthesis of the UCNP of the present invention.

[0189] Element [mol.%] Y, mol.% Yb, mol.% Tm, mol.% Nd, mol.% Theoretical 80.3 9.5 0.1 10 Experimental 81.5 9.0 0.1 9.4

[0190] To investigate the effect of temperature on UCNP emission, the photoluminescence (PL) emission spectra of UCNPs in a cyclohexane suspension for the same sample under room temperature (295 K) and post-thaw conditions were obtained with 808 nm laser excitation (0.5 W / cm²). 2 It was recorded under ), and the results are shown in Fig. 4d.

[0191] “Post-thaw” refers to a sample that was stored in a freezer at 193K for 10 minutes before PL measurement and then thawed at 295K. In the present invention, it was confirmed that the area of ​​the total PL emission peak increased by 2.45 times under post-thaw conditions, which can be interpreted as the effect of exposure to freezing.

[0192] When exposed to a low-temperature environment, cross-relaxation by photons is limited and excited states (e.g., 1 I6, 1 D2 and 1 The density of G4) was maximized, and radiative emission increased.

[0193] FIG. 6 is a schematic diagram of the upconversion process in the upconversion nanoparticles of the present invention. In FIG. 6, Yb 3+Nd at 808 nm excitation 3+ By transferring photons harvested from the photosensitizer, Tm 3+ It acts as an energy carrier that photoactivates emitter ions. Under NIR laser irradiation, Nd 3+ The ion harvests 808 nm photons and Yb by the cascade energy transfer mechanism shown in Fig. 6. 3+ ion( 2 F 5 / 2 → 2 F 7 / 2 Tm here energy through ) 3+ ion( 3 It was delivered to the bottom state of H6).

[0194] As a result, Tm 3+ The emitted ion is 289 nm 1 I6→ 3 H6, 344 nm 1 I6→ 3 F4, 361 nm 1 D2→ 3 H6, 450 nm 1 D2→ 3 F4 and 474 nm 1 G4→ 3 Visible emission was generated in H6.

[0195] The blue upconversion emission of UCNP after thawing could be easily observed with the naked eye under incandescent light on and off conditions (inset image in Fig. 4d). Therefore, it was confirmed that there are clear advantages to cryogenic control of upconversion brightness in order to develop a synergistic treatment method of cryotherapy and photodynamic therapy.

[0197] UCNP@SiO 2 Checking characteristics

[0198] Figure 3 is a low-resolution TEM image (scale bar: 1 μm) of UCNP@SiO2 nanoparticles to verify a uniform coating without severe aggregation. Synthesis was carried out under continuous sonication for the monocapsulation of UCNPs, which is intended to separate the UCNPs before encapsulation with a silica layer to prevent unwanted aggregation.

[0199] The successful encapsulation of UCNPs@SiO2 nanoparticles into a porous silica layer was confirmed by TEM as shown in Fig. 4e. Referring to Fig. 7, the geometric mean diameter of the UCNPs@SiO2 nanoparticles was 76.214 nm, which indicates that the average thickness of the silica layer surrounding the UCNPs is approximately 22 nm.

[0200] Figure 4f shows the Fast Fourier Transform (FFT) pattern of an HR-TEM image of UCNPs@SiO2 nanoparticles, through which information on the local diffraction pattern was obtained and confirmed that the hexagonal crystal structure of the UCNPs was maintained without damage.

[0201] Referring to Fig. 4f, the spacing between lattices was measured to be 5.112 Å (yellow dashed circle) and 5.192 Å (green dashed circle), respectively, corresponding to the hexagonal lattice parameters (5.16552 Å) of NaYF4, such as (-100) and (0-10). Referring to Fig. 4g, the X-ray diffraction (XRD) pattern of the total UCNPs@SiO2 powder showed the presence of broad and sharp peaks characteristic of amorphous silica, corresponding to the reference pattern of pure hexagonal phase β-NaYF4 (JCPDS Card 16-0334) without impurity peaks (e.g., cubic or NaF). These results confirmed that the hexagonal crystal structure of UCNPs is maintained even after the silica layer is formed under ultrasonic fracture synthesis conditions.

[0202] Figure 4h shows the HAADF-EDS elemental mapping of UCNPs@SiO2 nanoparticles, and it was confirmed that UCNPs@SiO2 was successfully fabricated by further verifying that representative elements such as Y, Yb, Tm, and Nd are uniformly distributed in the core structure and Si elements are uniformly distributed in the outermost shell. PL analysis results showed that the integrated PL intensity decreased by 22.9% after silica shell encapsulation, which may be due to absorption by the outer silica layer (Figure 8).

[0204] Verification of nanoplatform characteristics

[0205] Figure 9a is a schematic diagram for the fabrication of the UCNPs@SiO2-Ce6-HA nanoplatform. Before synthesizing UCNPs@SiO2 into Ce6 and HA, amine groups were introduced by surface modification with 3-aminopropyltriethoxysilane (APTES). This was confirmed by a change in zeta potential from a negative charge (-25.8 ± 0.84 mV) to a positive charge (38.75 ± 1.04 mV). Then, UCNPs@SiO2-NH2 was conjugated with Ce6 via an EDC / NHS chemical reaction.

[0206] The loading efficiency of Ce6 in the UCNPs@SiO2-Ce6 complex was estimated to be 29.2 wt.%, determined by the calibration curve of a standard Ce6 solution (Fig. 10). The zeta potential changed to a negative value of -29.84 ± 0.17 mV, reflecting the abundant carboxyl moiety on the surface of UCNPs@SiO2-Ce6. Subsequently, UCNPs@SiO2-Ce6 was conjugated with HA through the reaction of the carboxyl groups of UCNPs@SiO2-Ce6 with the amine groups of the HA-diaminobutane (DAB) conjugate.

[0207] 1¹H NMR analysis showed characteristic peaks observed in the δ1.9 (HA) and δ1.6 (DAB) regions occurring in the methyl moiety of HA and DAB with a DAB ratio of 32.25% (Fig. 11). The successful surface modification of UCNPs@SiO2 was confirmed by TEM images of the UCNPs@SiO2-Ce6 and UCNPs@SiO2-Ce6-HA nanoplatforms (Figs. 9b, 9c).

[0208] Due to the HA coating, the zeta potential shifted slightly to a negative value of -33.16 ± 0.03 mV, providing stable colloidal behavior in physiological environments. Dynamic light scattering (DLS) analysis revealed that the hydrodynamic size of the UCNPs@SiO2-Ce6-HA nanoplatform in phosphate-buffered saline (PBS) solution was approximately 177.7 nm, and it was confirmed to have a narrow size distribution (PDI: 0.2448, nearly monodisperse).

[0209] The evolution of molecular bonding during the fabrication of the UCNP@SiO2-Ce6-HA nanoplatform was evaluated using FT-IR spectroscopy (Fig. 9d). 1065 cm⁻¹ -1 and 799 cm -1 The peaks can be assigned to Si-O-Si asymmetric and symmetric stretching, respectively, which confirmed the presence of a silica shell.

[0210] Ce6 loading is 1538 cm -1 (COO- Stretching) and 1633 cm -1 It was confirmed as a new peak at (C=N stretching). Surface modification of UCNPs@SiO2-Ce6 with HA was 3291 cm⁻¹. -1 (OH Stretching), 2882 cm -1 (CH Stretching), 1394 cm -1 and 1376 cm -1 (CH bend) and 1045 cm -1 A peak of the absorption band was generated at (CO stretching).

[0211] The normalized absorbance profile showed a signal at 410 nm (Fig. 9e), which corresponds to the contribution of Ce6, confirming the successful fabrication of the UCNPs@SiO2-Ce6-HA nanoplatform.

[0212] To better understand the photochemical phenomena occurring under 808 nm NIR laser irradiation, singlet oxygen by UCNPs@SiO2-Ce6-HA nanoplatform ( 1 O2) generation was quantified by detecting it using probe molecules. The UCNPs of NaYF4:Yb,Tm@NaYF4:Nd converted the energy of several 808 nm NIR photons into high-energy UV photons, and by the Ce6 photosensitizer 3 Cytotoxicity of O2 1 It was possible to trigger photochemical conversion with O2.

[0213] In the present invention, a 1,3-diphenylisobenzofuran (DPBF) sensor is used to convert into colorless 1,3-diphenylisobenzoylbenzene 1 O2 was detected. 1 O2 generation can be quantitatively estimated from the gradual decrease in the characteristic absorption intensity around 410 nm in the UV-vis spectrum.

[0214] To investigate the temperature-responsive photo-singlet oxygen conversion, the DPBF decomposition rate was evaluated for 10 minutes under cryogenic (193 K) and room temperature (295 K) conditions as shown in Figs. 9f and 12. Surprisingly, 808 nm (0.5 W / cm²) 2 After being excited with an NIR laser in ), the UCNPs@SiO2-Ce6-HA nanoplatform was subjected to cryogenic conditions (slope 0.0555, R) as shown in Fig. 9g. 2 Significantly higher decomposition kinetics were exhibited at (=0.997). Relatively moderate photooxidation of DPBF was observed at room temperature (slope 0.0176, R 2 = 0.997).

[0215] As a result, when a near-infrared laser was irradiated for 10 minutes under cryogenic conditions, the UCNPs@SiO2-Ce6-HA nanoplatform 1 It was confirmed that the amount of O2 produced was improved by 3.15 times.

[0216] Based on these results, the in vitro biocompatibility and cellular uptake of the UCNPs@SiO2-Ce6-HA nanoplatform for Cryo-PDT cancer treatment were evaluated in the present invention. There was no severe toxicity caused by the UCNPs@SiO2-Ce6-HA nanoparticles in mouse fibroblast skin L929 cells and B16F10 melanoma cells (Fig. 13).

[0217] To visualize cell uptake, fluorescence imaging of UCNPs@SiO2-Ce6-HA was performed. Referring to Figure 14a, confocal laser scanning microscopy (CLSM) visualized the penetration of the UCNPs@SiO2-Ce6-HA nanoplatform into L929 and B16F10 cells. It was found that the red fluorescence signal intensity of UCNPs@SiO2-Ce6-HA increased 1.74-fold in B16F10 cells compared to L929 cells (Figure 14b). Using these characteristics, it was confirmed that UCNPs@SiO2-Ce6-HA selectively accumulates only in tumor cells while minimizing damage to normal cells.

[0218] Confocal laser scanning microscopy (CLSM) clearly visualized the effective infiltration of the UCNPs@SiO2-Ce6-HA nanoplatform into B16F10 melanoma cells by HA receptor-mediated endocytosis (Fig. 15a).

[0219] When HA receptors such as cluster determinant 44 (CD44) and lymphatic endothelial hyaluronic acid receptor-1 (LYVE-1) were inactivated by pre-culturing B16F10 cells with an excessive amount of HA, the cellular uptake of the UCNP@SiO2-Ce6-HA nanoplatform was significantly reduced due to competitive binding of HA.

[0220] These results showed that the UCNPs@SiO2-Ce6-HA nanoplatform was absorbed by HA receptor-mediated endocytosis.

[0221] Referring to Fig. 15a, the red fluorescence intensity in Ce6 around the nucleus increased dramatically in the absence of HA, reflecting the efficient cellular uptake of the UCNPs@SiO2-Ce6-HA nanoplatform by HA receptor-mediated endocytosis. These results confirmed the role of HA in the tumor-targeted accumulation of UCNPs@SiO2-Ce6-HA.

[0222] The antitumor therapeutic efficacy of the UCNPs@SiO2-Ce6-HA nanoplatform was evaluated by performing in vitro PDT. Intracellular singlet oxygen generation was visualized as a singlet oxygen-induced conversion of DCFH-DA into oxidized DCF with green fluorescence.

[0223] Figure 15b shows fluorescence microscopy images of the control, laser, and UCNPs@SiO2-Ce6-HA treatment groups, confirming that singlet oxygen production was absent or negligible as green fluorescence was indistinct. In contrast, vivid green fluorescence was observed in B16F10 cells cultured with UCNPs@SiO2-Ce6-HA followed by irradiation with an 808 nm laser (Laser + UCNPs@SiO2-Ce6-HA). The fluorescence intensity increased 113-fold compared to the other treatment groups, demonstrating efficient intracellular singlet oxygen production.

[0224] The in vitro NIR-mediated antitumor effect of the UCNPs@SiO2-Ce6-HA nanoplatform was quantitatively investigated using the CCK-8 assay. B16F10 cells were cultured for 24 hours with various concentrations of the UCNPs@SiO2-Ce6-HA nanoplatform ranging from 0 to 500 μg / mL. Subsequently, 0.5 W / cm² was applied to the cells. 2 An 808 nm NIR laser was irradiated for 10 minutes at a power density.

[0225] Referring to Figure 15c, the viability of B16F10 cells dropped to 22.3% at a concentration of 500 μg / mL upon 808 nm NIR irradiation. To further verify the results of the previous experiments, the in vitro antitumor therapeutic effect was visualized through simultaneous staining analysis of living and dead cells using Calcein AM and propidium iodide (PI).

[0226] In Figure 15d, no significant toxicity was observed in the control group, laser, and standard treatments using the UCNPs@SiO2-Ce6-HA nanoplatform, whereas in the treatment using the UCNPs@SiO2-Ce6-HA nanoplatform and an 808 nm laser (Laser + UCNPs@SiO2-Ce6-HA), green fluorescence (living cells) was almost non-existent and red fluorescence (dead cells) was strongly observed. These results confirmed that the UCNPs@SiO2-Ce6-HA nanoplatform exhibits high biocompatibility and near-infrared-mediated anticancer efficacy.

[0227] The in vitro transdermal delivery of the UCNPs@SiO2-Ce6-HA nanoplatform was evaluated using porcine skin, which has anatomical and physiological similarities to human skin.

[0228] HA is one of the essential biological components of the epidermis and dermis of human skin and is known to facilitate the transdermal delivery of drugs deep into the dermis.

[0229] Figure 15e shows confocal microscopy scanning images and depth profiles of the frozen sectioned porcine skin surface after topical in vitro administration of UCNPs@SiO2-Ce6 and UCNPs@SiO2-Ce6-HA nanoplatform solutions in PBS. The degree of skin penetration was monitored for 24 hours using the red fluorescence signal of the Ce6 photosensitizer in the skin. Figures 15e and 16 demonstrated a significant difference in skin penetration between the UCNPs@SiO2-Ce6-HA nanoplatform and the UCNPs@SiO2-Ce6 nanoparticles. It was confirmed that the HA-coated nanoparticles were evenly distributed in the deep layers of the skin as bright red fluorescence. The image in Figure 16 was obtained using the maximum intensity of a confocal microscopy scanning 405 nm UV laser module with a maximum output of 5 mW (scale bar: 100 μm).

[0230] The intensity gradually increased over time, reflecting that the UCNPs@SiO2-Ce6-HA nanoplatform demonstrated effective transdermal delivery through the stratum corneum and basal porcine skin layer. These results were also confirmed by the depth profile analysis shown in Figure 15f.

[0231] More specifically, the integrated red fluorescence intensity region in the epidermal layer increased 3.2-fold between 2 and 24 hours. As expected, the diffusion of UCNPs@SiO2-Ce6 was hindered by the skin barrier and accumulated mainly in the stratum corneum. These results were in good agreement with other reports elsewhere, demonstrating the excellent potential of the UCNPs@SiO2-Ce6-HA nanoplatform for effective transdermal delivery to deep skin layers and cancer cells. HA derivatives are also known to facilitate delivery to tumor cells with abundant HA receptors, such as CD44 and LYVE-1.

[0232] The in vivo antitumor effect of the UCNPs@SiO2-Ce6-HA nanoplatform was further confirmed in C57BL / 6 mice harboring B16F10 tumors. Cryotherapy was used on melanoma tumors to maximize the PDT efficacy of the UCNPs@SiO2-Ce6-HA nanoplatform. Mice harboring B16F10 tumors were randomly divided into the following seven groups: (1) PBS (Control), (2); UCNPs@SiO2-Ce6-HA, (3) Cryo, (4) Laser, (5) Cryo + UCNPs@SiO2-Ce6-HA, (6) Laser + UCNPs@SiO2-Ce6-HA (PDT), and (7) Cryo + Laser + UCNPs@SiO2-Ce6-HA (Cryo-PDT).

[0233] The tumor sites of groups 2, 5, 6, and 7 were topically treated with a UCNPs@SiO2-Ce6-HA solution (500 μg / mL, 50 μL) dispersed in PBS for 30 minutes. In cryo groups 3 and 5, the tumor sites were treated with liquid nitrogen for 10 minutes. Laser groups 4 and 6 used an 808 nm NIR laser (0.5 W / cm²). 2 , 10 minutes) was investigated.

[0234] Figure 17 shows a schematic diagram of the laser setup. The laser power density was adjusted using a power meter (843-R, Newport, USA). In Group 7, local administration of the UCNPs@SiO2-Ce6-HA solution (30 minutes) was followed by sequential 1-minute cryo and 1-minute laser cycle treatments (total 20 minutes).

[0235] Figure 18 shows the thermal mapping obtained from a single cryo / laser treatment cycle and the thermal imaging results captured during a cryo-PDT cycle including 1 minute cryo and 1 minute laser treatment. Each cryo cycle includes 30 seconds of freeze-thaw cycles. Thermal imaging was captured using a FLIR infrared camera, and the low-temperature sensitivity was limited to -20°C.

[0236] Figure 19a shows the temperature fluctuation curve. In vivo transdermal delivery of the UCNPs@SiO2-Ce6-HA nanoplatform was confirmed by confocal microscopy scanning images of frozen cancer tissue sections derived from the tumor area after local administration of PBS (Control), UCNPs@SiO2-Ce6, and UCNPs@SiO2-Ce6-HA solutions for 30 minutes.

[0237] Confocal microscopy scanning images showed vivid red fluorescence in deep regions, including the skin and tumor tissue beneath the skin, mainly from the UCNPs@SiO2-Ce6-HA nanoplatform in the entire observed cancer tissue (Fig. 19b). In contrast, UCNPs@SiO2-Ce6 showed strong fluorescence in the upper skin surface region of the tumor tissue, indicating that it did not penetrate well into the tumor located beneath the skin.

[0238] After treatment, mice were sacrificed and digital photographs of the collected tumors were taken as shown in Fig. 19c to clearly visualize the anti-tumor effect of the treatment group.

[0239] Tumor volume and mouse body weight were measured for 14 days. Figure 19d shows that mouse body weight did not decrease significantly during the treatment period, indicating that there was no severe toxicity caused by UCNPs@SiO2-Ce6-HA, laser, and Cryo treatments. The administered nanoparticle doses were found to induce negligible toxicity in vivo. As shown in Figure 19e, the UCNPs@SiO2-Ce6-HA and laser groups showed a degree of tumor inhibition similar to the control group. Slight tumor growth retardation was observed in the Cryo and Cryo + UCNPs@SiO2-Ce6-HA groups, which had similar tumor growth inhibition rates of approximately 17%.

[0240] In particular, statistically different tumor growth was inhibited in mice treated in the PDT group. The only PDT (laser + UCNPs@SiO2-Ce6-HA) treatment inhibited tumor growth by more than 55% over 14 days. However, the Cryo-PDT group (Cryo + Laser + UCNPs@SiO2-Ce6-HA) achieved the greatest tumor volume regression of 79%. Successful Cryo-PDT treatment can be attributed to continuous Cryo and laser treatment cycles that maintain cooling of the tumor site for the effective upconversion of UCNPs.

[0241] Finally, tumor sections were stained with hematoxylin and eosin (H&E) and exposed to terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) to examine tumor histology and apoptosis. Figure 19f shows representative histological images of tumors after various treatments.

[0242] In particular, tumor histological structure and cell viability did not change significantly in the control, UCNPs@SiO2-Ce6-HA, and laser groups. Slight morphological changes and apoptosis were observed in the Cryo and Cryo + UCNPs@SiO2-Ce6-HA groups. Consistent with tumor growth, tumors treated with the Cryo-PDT treatment group (Cryo + Laser + UCNPs@SiO2-Ce6-HA) exhibited the most severe tissue damage and distinct apoptosis and necrosis in H&E stained images.

[0243] TUNEL analysis also supported the antitumor effect through apoptosis exhibiting a strong green fluorescence signal in key sites compared to PDT treatment alone without the cryo-effect. Taken together, the remarkable synergistic therapeutic efficacy of the Cry-PDT therapy of the present invention against cutaneous melanoma was successfully demonstrated.

[0245] Although the present invention has been described above with reference to embodiments, the present invention is not limited by the embodiments disclosed in this specification, and it is obvious that various modifications can be made by a person skilled in the art within the scope of the technical concept of the present invention. Furthermore, even if the effects of the configuration of the present invention were not explicitly described while describing the embodiments of the present invention above, it is natural to acknowledge that the effects predictable by said configuration should also be recognized.

Claims

Claim 1 A nanoplatform comprising a core-shell structured upconversion nanoparticle comprising a core made of NaYF4:Yb, Tm; and a shell made of NaYF4:Nd surrounding the core; a porous shell made of SiO2 on the shell made of NaYF4:Nd of the upconversion nanoparticle, wherein the porous shell is loaded with at least one photosensitive agent, and hyaluronic acid is bound to the porous shell loaded with the photosensitive agent. Claim 2 delete Claim 3 In claim 1, the porous shell comprises chlorine e6, zinc phthalocyanine (ZnPC), aminolevulinic acid (ALA), methyl aminolevulinate, temoporfin, phthalocyanine, protoporhap IX (PpIX), Allumera™, Cevira™, Hexvix™, porfimer sodium, verteporfin, δ-aminolevulinic acid or 5-aminolevulinic acid, temoporfin, methyl aminolevulinate, hexaminolevulinate hydrochloride, talaporfin, motexafin lutetium, and 2-(1-hexyloxyethyl)-2-devinyl A nanoplatform loaded with at least one photosensitizer selected from the group consisting of pyropheophorbide-a (2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a), Photosens™, rostaporfin, BF-200 ALA, tetraphenyl chlorin disulfonate, and azadipyrromethene. Claim 4 delete Claim 5 A nanoplatform according to claim 1, wherein the size of the upconversion nanoparticles is 10 to 100 nm. Claim 6 A nanoplatform according to claim 1, wherein the thickness of the porous shell is 1 to 50 nm. Claim 7 A pharmaceutical composition for the prevention or treatment of cancer comprising a nanoplatform according to claim 1. Claim 8 A pharmaceutical composition for the prevention or treatment of cancer according to claim 7, wherein the cancer is one or more selected from the group consisting of melanoma, lung cancer, head and neck cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, sarcoma, renal cell carcinoma, stomach cancer, esophageal cancer, anal canal cancer, cholangiocarcinoma, pancreatic cancer, liver cancer, cervical cancer, acute myeloid leukemia, chronic myeloid leukemia, skin cancer, lymphoma, thyroid cancer, bone marrow cancer, endometrial cancer, and brain tumor. Claim 9 A pharmaceutical composition for the prevention or treatment of cancer, wherein, in claim 7, the treatment is intended for use in a treatment selected from the group consisting of photodynamic therapy, cryotherapy, and combinations thereof. Claim 10 A food composition for preventing or improving cancer, comprising a nanoplatform according to claim 1. Claim 11 A food composition for preventing or improving cancer, wherein the cancer is one or more selected from the group consisting of melanoma, lung cancer, head and neck cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, sarcoma, renal cell carcinoma, stomach cancer, esophageal cancer, anal canal cancer, cholangiocarcinoma, pancreatic cancer, liver cancer, cervical cancer, acute myeloid leukemia, chronic myeloid leukemia, skin cancer, lymphoma, thyroid cancer, bone marrow cancer, endometrial cancer, and brain tumor.

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