Pharmaceutical composition comprising upconversion nanoparticles for preventing or treating cancer

Abstract

The present invention relates to a nano-platform comprising core-shell structured upconversion nanoparticles, a composition comprising same, and a treatment method, the nano-platform comprising: a core formed of NaYF4:Yb, Tm through single-step synthesis; and a shell, which encompasses the core and is formed of NaYF4:Nd.

Description

Pharmaceutical composition for preventing or treating cancer comprising upconversion nanoparticles

[0001] The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising upconversion nanoparticles.

[0002]

[0003] Cryoablation and cryosprayer are low-temperature therapeutic approaches for various human cancers that involve lethal repeated freezing and thawing of cancer cells.

[0004] Despite its long history as a treatment, this treatment falls short of completely blocking cancer progression. Recently, nanoparticles have been reported to hold great promise for enhancing the effectiveness of cancer treatment.

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

[0006] Kwak et al. reported the incorporation of thermally conductive inorganic nanoparticles such as magnesium oxide, gold, silver, and iron oxide nanoparticles into cryogenic therapeutics to maximize the degree of intracellular freezing, drug delivery, and image guidance.

[0007] However, clinical applications are still limited because intravenously injected nanoparticles have difficulty reaching the target tumor site, resulting in nonspecific biodistribution and unsatisfactory therapeutic effects.

[0008] Therefore, research on the efficacy of low-temperature-responsive nanomaterials and alternative delivery methods with optimal administration plans is necessary for a breakthrough in cryotherapy.

[0009]

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

[0011] In addition, an object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, including a nanoplatform that can be used for cryotherapy (Cryo) and photodynamic therapy (PDT) treatments.

[0012] In addition, an object of the present invention is to provide a food composition for preventing or improving cancer, which comprises the nanoplatform.

[0013] In addition, an object of the present invention is a method for treating cancer, comprising a step of administering the nanoplatform to a subject in a therapeutically effective amount.

[0014] The problems to be solved by the present invention are not limited to the problem(s) mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0015]

[0016] To achieve the above object, the present invention provides a nanoplatform including an upconversion nanoparticle having a core-shell structure, including a core composed of NaYF4:Yb, Tm; and a shell composed 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 porous shell is composed of chlorine e6, zinc phthalocyanine (ZnPC), aminolevulinic acid (ALA), methyl aminolevulinate, temporfin, phthalocyanine, protoporhap IX (PpIX), Allumera TM , Cevira TM , Hexvix TM, porfimer sodium, verteporfin, δ-aminolevulinic acid or 5-aminolevulinic acid, temoporfin, methyl aminolevulinate, hexaminolevulinate hydrochloride, talaporfin, motexafin lutetium, 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a, Photosens TM , rostaporfin, BF-200 ALA, tetraphenyl chlorin disulfonate, and azadipyrromethene.

[0019] The above porous shell may be bound with hyaluronic acid.

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

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

[0022]

[0023] In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the nanoplatform.

[0024] The cancer may be at least one 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, gastric cancer, esophageal cancer, anal tract cancer, biliary tract cancer, 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 for use in a treatment selected from the group consisting of photodynamic therapy, cryotherapy and combinations thereof.

[0026]

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

[0028] The cancer may be at least one 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, gastric cancer, esophageal cancer, anal tract cancer, biliary tract cancer, 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.

[0029]

[0030] The present invention also provides a method for treating cancer, comprising administering the nanoplatform to a subject in a therapeutically effective amount.

[0031]

[0032] The nanoplatform comprising the upconversion nanoparticles of the present invention can be used in combination treatment with cryotherapy and photodynamic therapy (PDT) to more effectively treat skin melanoma in particular.

[0033]

[0034] 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 detailed description of the present invention or the composition of the invention described in the claims.

[0035]

[0036] Figure 1 is a schematic diagram of a nanoplatform comprising core-shell structured upconversion nanoparticles for synergistic anticancer effects of percutaneous Cryo-PDT treatment for cutaneous melanoma.

[0037] Figure 2 schematically illustrates the reaction time optimization for size uniformity of core(NaYF4:Yb,Tm)@shell(NaYF4:Nd) upconversion nanoparticles manufactured in a single step via the Ostwald ripening method.

[0038] Figure 3 is a low-resolution TEM image (scale bar: 1 μm) of UCNP@SiO2 nanoparticles to verify uniform coating and dispersion without severe agglomeration.

[0039] Figure 4A is a schematic diagram for the single-step synthesis of UCNPs comprising NaYF4:Yb,Tm@NaYF4:Nd(core@shell).

[0040] Figure 4B is a TEM image of synthesized UCNPs (scale bar: 50 nm).

[0041] Figure 4C shows the SAED pattern of UCNPs (scale bar: 5 nm -1 ) is the result.

[0042] Figure 4D is the photoluminescence emission spectrum of the UCNPs solution in cyclohexane after thawing and storage at room temperature (808 nm NIR laser irradiation, 10 W / cm 2 ), the inset images here are digital images of the visually observable blue upconversion emission of a UCNP solution after dissolution in cyclohexane at room temperature under 808 nm laser irradiation with the light on and off.

[0043] Figure 4E is a TEM image of UCNPs@SiO2 (scale bar: 50 nm).

[0044] Figure 4F is an FFT obtained from the HR-TEM image of UCNPs@SiO2.

[0045] Figure 4G shows the X-ray diffraction pattern of the UCNPs@SiO2 sample and the reference pattern (JCPDS 16-0334) of hexagonal β-NaYF4.

[0046] Figure 4H shows the HAADF-STEM image and STEM-EDS elemental mapping results (scale bar: 25 nm) of the UCNPs@SiO2 sample.

[0047] Figure 5 shows the HAADF-STEM image and STEM-EDS elemental mapping results (scale bar: 25 nm) of UCNPs.

[0048] Figure 6 is a schematic diagram of the upconversion process in the upconversion nanoparticles of the present invention.

[0049] The left side of Fig. 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) is the result, and the right side of Fig. 7 is a high-resolution TEM image of UCNPs@SiO2 (scale bar: 50 nm).

[0050] Figure 8 shows the photoluminescence emission profiles of UCNPs before and after silica shell encapsulation under 808 nm laser excitation.

[0051] Figure 9A is a schematic diagram for preparing UCNP@SiO2-Ce6-HA nanoplatform.

[0052] Figure 9B is a TEM image of UCNPs@SiO2-Ce6 (scale bar: 50 nm).

[0053] Figure 9C is a TEM image of UCNPs@SiO2-Ce6-HA (scale bar: 50 nm).

[0054] Figure 9D is the FT-IR spectra of UCNPs@OA, UCNPs@SiO2-NH2, UCNPs@SiO2-Ce6, and UCNPs@SiO2-Ce6-HA.

[0055] Figure 9E is the normalized absorbance spectra of Ce6, UCNPs@SiO2, UCNPs@SiO2-Ce6, and UCNPs@SiO2-Ce6-HA.

[0056] Figure 9F is the singlet oxygen production profile of UCNPs@SiO2-Ce6-HA under cryogenic conditions under 808 nm NIR laser irradiation using a DPBF sensor.

[0057] Figure 9G shows the results comparing the photodegradation rate of DPBF by UCNPs@SiO2-Ce6-HA under cryogenic (blue) and room temperature (red) conditions.

[0058] Figure 10 is a Ce6 standard curve including five data points, based on the absorption spectra of Ce6 solutions in dimethylformamide at various concentrations.

[0059] Figure 11 shows the chemical structure of the HA-DAB conjugate and its structure in deuterium oxide. 1 The H NMR spectrum is shown. Here, m and n are integers independently selected from 16 to 2500.

[0060] Figure 12 shows the singlet oxygen production profile results measured at room temperature of the UCNPs@SiO2-Ce6-HA nanoplatform under 808 nm laser irradiation using a DPBF sensor.

[0061] Figure 13 shows 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.

[0062] Figure 14A shows the results of cell uptake after treating L929 and B16F10 cells with UCNPs@SiO2-Ce6-HA nanoplatform and various substances at a concentration of 500 μg / mL for 4 hours after incubation (scale bar: 150 μm).

[0063] Figure 14B is the quantitative fluorescence intensity result of Figure 14A (n=3, **P <0.01, ***P <0.001).

[0064] Figure 15A shows confocal microscopy results (blue: DAPI, red: Ce6, scale bar: 20 μm) of B16F10 cells after treatment with Ce6 and UCNPs@SiO2-Ce6-HA with or without HA pre-incubation.

[0065] Figure 15B shows the various treatments: untreated (control), 808 nm laser only, UCNPs@SiO2-Ce6-HA only, and 808 nm laser and UCNPs@SiO2-Ce6-HA together (laser irradiation was 0.5 W / cm 2 This is the result of intracellular singlet oxygen detection in B16F10 cells after 10 minutes of irradiation with a 10-minute time point, and the green fluorescence of DCF reflects the presence of singlet oxygen (scale bar: 150 μm).

[0066] Figure 15C shows the cell viability (mean ± SD, n = 3, ***P < 0.001) results of B16F10 cells cultured in various concentrations of UCNPs@SiO2-Ce6-HA (control, 10, 50, 100, 200, 500 μg / mL) with or without 808 nm laser irradiation (w / o) to determine the photodynamic therapy efficiency in vitro.

[0067] Figure 15D shows the various treatments: untreated (control), 808 nm laser only, UCNPs@SiO2-Ce6-HA only, and 808 nm laser and UCNPs@SiO2-Ce6-HA together (laser irradiation was 0.5 W / cm 2After 10 minutes of investigation by the century, the Live / dead analysis of B16F10 cells was observed under a fluorescence microscope (green: calcein-AM, red: PI, scale bar: 300 μm), where green or red fluorescence represents live cells or dead cells, respectively.

[0068] Figure 15E shows photographs (scale bar: 50 μm) taken using a confocal laser scanning microscope after collecting skin tissue using a freezing microtome 2, 4, 8, 12, and 24 hours after topical application of UCNPs@SiO2-Ce6 and UCNPs@SiO2-Ce6-HA solutions (500 μg / mL) dispersed in PBS to the surface of pig skin.

[0069] Figure 15F shows the results of observing the depth of penetration of the nanoplatform into skin tissue (SC: stratum corneum, Epi.: epidermis) from pig skin treated with UCNPs@SiO2-Ce6-HA after 2, 4, 8, 12, and 24 hours.

[0070] Figure 16 shows photographs of skin tissue collected with a freezing microtome 2, 4, 8, 12, and 24 hours after topical application of a solution of UCNPs@SiO2-Ce6 (500 μg / mL) dispersed in PBS to pig skin, observed using a confocal laser scanning microscope (scale bar: 100 μm).

[0071] Figure 17 is a schematic diagram of the laser setup.

[0072] Figure 18 shows the thermal imaging results captured 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.

[0073] Figure 19A shows the temperature change profile results for Cryo-PDT treatment cycles, each cycle includes 1 minute of Cryo and 1 minute of Laser (10 cycles in total), and according to the FLIR infrared camera user manual, the lowest temperature measurement range is limited to -20°C.

[0074] Figure 19B shows 500 μg / mL of UCNPs@SiO2-Ce6 and UCNPs@SiO2-Ce6-HA solutions dispersed in PBS. (scale bar: 150 μm) This is an image of a confocal laser scanning microscope image of a frozen section of melanoma tumor tissue after topical treatment in vivo at a concentration of 100 μg / ml. The dotted line indicates the skin / tumor boundary.

[0075] Figure 19C is a representative photograph of melanoma tumor tissues harvested 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.

[0076] Figure 19D is a graph of relative weight change.

[0077] Figure 19E is the relative tumor growth curve after various treatments for 14 days, error bars represent the standard deviation (SD) of four (n=4) individual sample measurements, and statistical significance was assessed using ANOVA (**P < 0.01 and ***P < 0.001).

[0078] Figure 19F shows the results of H&E and TUNEL staining (scale bar: 150 μm) of tumor tissue after various treatments.

[0079]

[0080] The terms used in this specification have been selected from widely used, current terms, taking into account their functions within the present invention. However, these terms may vary depending on the intentions of those skilled in the art, precedents, or the emergence of new technologies. Furthermore, in certain cases, the applicant may arbitrarily select terms, in which case their meanings will be described in detail in the relevant description of the invention. Therefore, the terms used in this specification should not be defined simply as names, but rather based on their inherent meanings and the overall content of the present invention.

[0081] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Commonly understood terms should be interpreted as having the same meaning within the context of the relevant technology, and unless explicitly defined herein, they shall not be construed in an idealized or overly formal sense.

[0082] Numerical ranges are inclusive of the numerical values ​​defined herein. Throughout this specification, any maximum numerical limitation given includes any lower numerical limitation, as if that lower numerical limitation were explicitly stated. Throughout this specification, any minimum numerical limitation given includes any higher numerical limitation, as if that higher numerical limitation were explicitly stated. Throughout this specification, any numerical limitation given includes any better numerical range within that broader numerical range, as if that narrower numerical limitation were explicitly stated.

[0083]

[0084] Hereinafter, the present invention will be described in detail.

[0085] In one embodiment of the present invention, a nanoplatform comprising upconversion nanoparticles having a core-shell structure is provided.

[0086] In one embodiment, a schematic diagram of a nanoplatform comprising core-shell structured upconversion nanoparticles for combined treatment of cryotherapy and photodynamic therapy (also 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 synergistic anticancer effects of percutaneous Cryo-PDT therapy for skin melanoma.

[0087] In one embodiment, the core-shell structured upconversion nanoparticles (also referred to as "UCNPs" or "UCNPs") may comprise a core composed of NaYF4:Yb, Tm; and a shell composed of NaYF4:Nd surrounding the core; wherein the core and shell are composed of a compound of formula M comprising a lanthanide ion. 1 M 2 It can be formed from a composition comprising a rare earth metal fluoride according to the formula F4. In the above formula, M 1 It may contain 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). M 2 is a rare earth element and may include at least one element 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 chemical formula may be included in an amount of 0 mol% to 99 mol%.

[0088] In one embodiment, the upconversion nanoparticles may be prepared through a single-step (in-situ) synthesis.

[0089] In one embodiment, the UCNPs can enhance upconversion luminescence efficiency due to increased near-infrared absorption at 808 nm.

[0090] In one embodiment, the nanoplatform may further comprise 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 a PDT photosensitizer described below.

[0091] In one embodiment, the porous shell may be loaded with a PDT photosensitizer, for example, chlorin 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. At least one photosensitizer selected from the group consisting of lutetium, 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a, Photosens™, rostaporfin, BF-200 ALA, tetraphenyl chlorin disulfonate and azadipyrromethene may be loaded, and upon irradiation with a red laser, singlet oxygen ( 1 02) can be generated. The viability of cancer cells is reduced by the singlet oxygen. The photosensitizer can be selected as an appropriate photosensitizer depending on the purpose of the invention.

[0092] In one embodiment, the porous shell may be bound with hyaluronic acid. The binding of hyaluronic acid to the porous shell may enable the nanoplatform to be transdermally delivered into cancer tissue and further stabilized in a physiological environment.

[0093] The above hyaluronic acid can be bonded or combined to the porous shell using EDC-NHS coupling.

[0094] In one embodiment, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is combined with N-hydroxysuccinimide (NHS) or sulfo-NHS to mean a coupling reaction used for immobilization, etc.

[0095] The size of the above upconversion nanoparticles may be 10 to 100 nm, specifically 20 to 40 nm, and more specifically 30 to 35 nm.

[0096] The thickness of the porous shell may be 1 to 50 nm.

[0097]

[0098] In one embodiment of the present invention, a pharmaceutical composition for preventing or treating cancer comprising the nanoplatform is provided.

[0099] The above nanoplatform is identical to the nanoplatform described above, and any content that overlaps with that described in the above nanoplatform will not be described again.

[0100] The term "photodynamic therapy (PDT)" as used herein refers to a method in which a photosensitizer is activated by light and then chemically reacts with molecular oxygen to produce singlet oxygen (SOO). 1 It refers to a next-generation treatment that generates singlet oxygen (O2) and selectively destroys the targeted cells or tissues with this singlet oxygen.

[0101] The term "cryotherapy" as used herein refers to a treatment method that selectively destroys or treats abnormal skin tissue or induces changes in local immune responses by rapidly freezing skin lesions using a very low temperature medium.

[0102] The term "cancer" as used herein is a general term for a disease caused by cells that have an aggressive characteristic in which cells divide and grow while ignoring normal growth limits, an invasive characteristic in which cells invade surrounding tissues, and a metastatic characteristic in which cells spread to other parts of the body. The cancer may be, for example, 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, gastric cancer, esophageal cancer, anal tract cancer, biliary tract cancer, pancreatic cancer, liver cancer, cervical cancer, acute myelogenous leukemia, chronic myelogenous leukemia, skin cancer, lymphoma, thyroid cancer, bone marrow cancer, endometrial cancer, and brain tumor.

[0103] In one embodiment, the cancer may be melanoma.

[0104] In one embodiment, the treatment may be for use in a treatment selected from the group consisting of photodynamic therapy, cryotherapy, and combinations thereof.

[0105] In the above cancer, the nanoplatform of the present invention can exhibit medically useful effects such as disease prevention, improvement, and treatment, and this is supported by the specification, examples, and experimental examples of the present application, and a pharmaceutical composition containing it as an effective ingredient can be provided.

[0106] Meanwhile, depending on the dosage and method of use of the pharmaceutical composition of the present invention, the content of the nanoplatform, which is an effective ingredient, can be appropriately adjusted and used according to the selection of a person skilled in the art.

[0107] In one embodiment, the pharmaceutical composition may comprise the nanoplatform in an amount of 0.01 to 50 wt%, preferably 0.1 to 25 wt%, and more preferably 0.1 to 10 wt%, based on the total weight of the entire composition.

[0108] In one embodiment, the nanoplatform may be included solely in the pharmaceutical composition, or may further include other pharmacologically acceptable carriers, excipients, diluents, etc.

[0109] The above excipients may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, moisturizers, film-coating materials, and controlled-release additives.

[0110] In one embodiment, the pharmaceutical composition may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, ellipsoids, emulsions, suspensions, alcohols, troches, air fresheners, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, ointments, lotions, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, according to a conventional method, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasmas. there is.

[0111] In one embodiment, carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0112] When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0113] Excipients for the above tablets, powders, granules, capsules, pills, and troches include 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, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC), HPMC 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, Primogel, etc.; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, and binders such as hydroxypropyl methylcellulose, corn starch, agar powder, methyl cellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Disintegrants such as carboxymethylcellulose calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, baking soda, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium dentata, kaolin, petrolatum, sodium stearate, cacao butter, 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 silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid can be used.

[0114] In one embodiment, additives of the liquid formulation may include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc.

[0115] The syrup may contain a solution of white sugar, other sugars, or sweeteners, and may also contain a fragrance, colorant, preservative, stabilizer, suspending agent, emulsifier, or viscosity modifier, as needed.

[0116] Purified water may be used in the above-mentioned emulsion, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0117] In one embodiment, suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, HPMC 2910, etc. may be used as suspending agents, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

[0118] In one embodiment, the injection comprises: a solvent such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, benzene benzoate; a solubilizer such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nitrile acid amide, hexamine, dimethylacetamide; a buffer such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumins, peptones, 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; oxidizing agents such as sodium bisulfite 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid, and acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium cis-methylenediamine, sodium alginate, Tween 80, and aluminum monostearate.

[0119] Solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules. These solid dosage forms may be prepared by mixing one or more excipients, such as starch, calcium carbonate, sucrose or lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

[0120] Liquid preparations for oral administration include suspensions, suspensions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives.

[0121] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0122] In one embodiment, the suppository comprises cocoa butter, lanolin, withepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, ranette wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydroxocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Mechanisms such as Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppository type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecovi (W, R, S, M, Fs), Tezester triglyceride basis (TG-95, MA, 57) can be used.

[0123] 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. The effective dosage level can be determined according to the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the excretion rate, the treatment period, the concurrently used drugs, and other factors well known in the medical field, and can be administered once or in several divided doses. As an active ingredient of the pharmaceutical composition, it can be administered to mammals, including humans, preferably in an amount of 0.001 to 100 mg / kg body weight, preferably 0.01 to 35 mg / kg body weight, once a day or in divided doses, via the oral or parenteral route.

[0124] In one embodiment, the pharmaceutical composition may be administered as an individual therapeutic agent or in combination with another therapeutic agent, administered sequentially or simultaneously with conventional therapeutic agents, or administered singly or in multiple doses. Considering all of the above factors, it is important to administer an amount that achieves maximum effect with the minimum amount possible without causing side effects. This can be readily determined by those skilled in the art.

[0125] In one embodiment, the pharmaceutical composition may be administered to a subject by various routes. All modes of administration are contemplated, including oral ingestion, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosa administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, and transdermal administration.

[0126] In one embodiment, the pharmaceutical composition is determined based on the type of drug as the active ingredient along with several related factors such as the disease to be treated, the route of administration, the patient's age, sex, weight, and severity of the disease.

[0127]

[0128] In one embodiment of the present invention, a method for treating cancer is provided, comprising administering a therapeutically effective amount of the nanoplatform to a subject.

[0129] The above nanoplatform is identical to the nanoplatform described above, and any content that overlaps with that described in the above nanoplatform will not be described again.

[0130] The above cancer is the same as the cancer of the pharmaceutical composition for preventing or treating cancer described above, and the overlapping content will not be described again.

[0131] The above treatment method may further include a step of identifying a patient in need of prevention or treatment of the above-described cancer prior to the above-described administration step.

[0132] The above "therapeutically effective amount" means the amount of an effective ingredient for mammals that is effective in preventing or treating cancer, and the above therapeutically effective amount can be adjusted according to various factors including the type of disease, the severity of the disease, the types and contents of the effective ingredient and other ingredients contained in the composition, the type of formulation, and the patient's age, weight, general health, sex, and diet, administration time, administration route, and blood clearance of the composition, treatment period, and concurrently used drugs.

[0133] In one embodiment, the nanoplatform may be administered orally or parenterally once a day or in divided doses in an amount of 0.001 to 100 mg / kg body weight, preferably 0.01 to 35 mg / kg body weight, on a daily basis.

[0134] The above “subject” may mean, but is not limited to, a mammal such as a human or non-human primate, mouse, dog, cat, horse, cow, etc.

[0135]

[0136] In one embodiment of the present invention, a food composition for preventing or improving cancer, comprising the nanoplatform, is provided.

[0137] The above nanoplatform is identical to the nanoplatform described above, and any content that overlaps with that described in the above nanoplatform will not be described again.

[0138] The cancer may be at least one 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, gastric cancer, esophageal cancer, anal tract cancer, biliary tract cancer, 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.

[0139] In one embodiment, when the nanoplatform is used as a food additive, the nanoplatform may be added as is or used together with other foods or food ingredients, and may be used appropriately according to a conventional method. The amount of the active ingredient mixed may be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing a food or beverage, the nanoplatform of the present invention may be added in an amount of 15 wt% or less, or 10 wt% or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may also be used in an amount above the above range.

[0140] There are no specific restrictions on the types of the above foods. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and all health functional foods in the conventional sense are included.

[0141] In one embodiment, the health beverage composition may contain various flavoring agents or natural carbohydrates as additional ingredients, just like a regular beverage. The natural carbohydrates mentioned above include 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 a sweetener, a natural sweetener such as thaumatin or stevia extract, or a synthetic sweetener such as saccharin or aspartame may be used. The proportion of the natural carbohydrate is generally about 0.01-0.20 g, or about 0.04-0.10 g, per 100 mL of the composition of the present invention.

[0142] 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 colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients may be used independently or in combination. The proportion of these additives is not particularly critical, but is typically selected within the range of 0.01 to 0.20 parts by weight per 100 parts by weight of the composition of the present invention.

[0143] In one embodiment, "subject" means a subject in need of treatment for a disease, and more specifically, a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow.

[0144] In one embodiment, “administering” means providing a composition of the invention to a subject by any suitable means.

[0145] In one embodiment, “prevention” means any action that inhibits or delays the onset of a target disease, “treatment” means any action that improves or beneficially changes a target disease and its accompanying metabolic abnormality symptoms by administering a pharmaceutical composition according to the present invention, and “improvement” means any action that reduces a parameter related to a target disease, for example, the severity of a symptom, by administering a composition according to the present invention.

[0146]

[0147] The above description has explained the technical idea of ​​the present invention using one embodiment. Those skilled in the art will appreciate that various modifications and variations can be made without departing from the essential characteristics of the present invention. Therefore, the embodiments described in the present invention are not intended to limit the technical idea of ​​the present invention, but rather to explain it, and the scope of the technical idea of ​​the present invention is not limited by these embodiments. The scope of protection of the present invention should be interpreted by the claims, and all technical ideas within a scope equivalent thereto should be interpreted as being included within the scope of the rights of the present invention.

[0148] <Example>

[0149] ingredient

[0150] 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), tetraethoxy silane (TEOS, 99.9%) and 1,3-diphenylisobenzofuran (97%) were all purchased from Alfa Aesar.

[0151] Methanol (99.9%), absolute ethanol (99.9%), sodium hydroxide pellets (>97%), and sodium chloride were purchased from Daejung Chemicals & Metals. Ammonium nitrate (≥98%), cetyltrimethylammonium bromide (CTAB, ≥98%), (3-aminopropyl)triethoxysilane (APTES, 99%), and 4',6-diamidino-2-phenylindole (DAPI) were purchased from Sigma Aldrich.

[0152] 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 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. Chlorin e6 was obtained from MedChem Express LLC. 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) was purchased from Cayman Chemical Company.

[0153] 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.

[0154]

[0155] Single-step synthesis of upconversion nanoparticles (referred to as "UCNPs" or "UCNPs")

[0156] UCNPs were synthesized using the Ostwald ripening method using high-temperature co-precipitation.

[0157] Specifically, 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); shell solution (hereinafter referred to as "solution B"): Y(CH3COO)3 (0.2 M, 4 mL), Nd(CH3COO)3 (0.2 M, 1.0 mL) were prepared in 250 mL three-necked round flasks, respectively. The round flasks were heated to 110°C for 10 min to evaporate water, and then a mixture of OA (6.0 mL) and ODE (15.0 mL) was added to solution A and solution B, respectively.

[0158] Then, the flask containing solution A was connected to a Schlenk line for gas removal and nitrogen purge. The flask containing solution A was maintained at 150°C for 30 min under a weak flow of nitrogen (N2) or nitrogen (N2) and hydrogen (H2<10%) to completely dissolve the lanthanide salt in the OA and ODE mixture. 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, then heated to 110°C under a flow of nitrogen (N2) or nitrogen (N2) and hydrogen (H2) to evaporate the methanol, and the gas was removed for 20 min. After the methanol was completely removed, the flask containing solution A was heated at 300°C in a nitrogen (N2) atmosphere for 1 h. Afterwards, the flask containing solution B was precipitated, degassed, and heated to 150°C (in the presence of N2), and then injected into the flask containing solution A at 300°C, and the reaction was further carried out for 1 h, 1.5 h, and 2 h for size focusing (Fig. 2, where the particle size distribution count N = 300, and the scale bar in the TEM image represents 100 nm). A double nitrogen balloon system was used to prevent overpressure in the reactor system. After cooling to room temperature, acetone was added to precipitate UCNPs, which were then extracted by centrifugation. After washing with acetone several times, they were finally redispersed in cyclohexane (20 mL) for further use.

[0159]

[0160] Synthesis of UCNPs coated with mesoporous silica (referred to as "UCNP@SiO2" or "UCNPs@SiO2")

[0161] First, 4 mL of 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 gentle heating at 50 °C for 2 h in a bath-sonicator. Subsequently, 20 mL of DI water was added to the dispersion, the pH was adjusted to 10.0 with 0.1 M NaOH solution, and then the dispersion was further homogenized by ultrasonication for 30 min. Next, TEOS and absolute ethanol were well mixed in a volume ratio of 1:4 and then injected dropwise into the solution while continuously applying ultrasonication to prevent aggregation (Fig. 3).

[0162] The solution was then kept at 30°C for 24 h with vigorous stirring. The synthesized UCNPs@SiO2 was obtained by centrifugation at 8000 rpm for 20 min and washed several times with ethanol. The CTAB surfactant was removed by ion exchange. The UCNPs@SiO2 was transferred into a flask containing 0.3 g NH4NO3 dissolved in 40 mL of ethanol. The flask was placed under ultrasonication at 50°C for 1 h. The CTAB extraction process was repeated twice. Next, the obtained UCNPs@SiO2 was grafted with amine groups to further load therapeutic ingredients.

[0163] First, UCNP@SiO2 was redispersed in a solvent mixture of ethanol / water (95:5 by weight), 100 μL APTES was added, and the mixture was vigorously stirred at 70 °C for 2 h under reflux. Finally, UCNP@SiO2-NH2 was separated by centrifugation at 8000 rpm for 20 min and redispersed in 4 mL DI water for further use.

[0164] For bioimaging purposes, UCNP@SiO2-NH2 nanoparticles were labeled with FITC photosensitizer by simple stirring overnight. The labeling transformation was evidenced by a change in sample color from white to yellow.

[0165]

[0166] Loading Ce6 photosensitive agent onto UCNPs coated with mesoporous silica (referred to as "UCNP@SiO2-Ce6" or "UCNPs@SiO2-Ce6")

[0167] 3 mg of Ce6 was dispersed in 4 mL of DI water, and its carboxyl group was activated by EDC / NHS reaction. Then, 4 mL of UCNPs@SiO2-NH2 (10 mg) dispersed in DI water was added to the solution containing Ce6, and ultrasonication was performed for 10 min to form a uniform dispersion.

[0168] To avoid interference from ambient light, the dispersion was then placed in an amber glass vial and stirred vigorously at room temperature. After 24 h of reaction, the solution was centrifuged at 8,000 rpm for 20 min to precipitate UCNPs@SiO2-Ce6. The solution was washed three times with DI water to remove excess Ce6, and the absorption spectrum of the collected supernatant was measured to quantify unreacted Ce6.

[0169] The loading capacity was calculated based on the absorbance calibration plot of a known Ce6 concentration solution. The loading capacity was confirmed to be 29.2 wt% based on the calibration curve in Figure 10. Finally, the resulting UCNPs@SiO2-Ce6 were freeze-dried and stored at -20°C in a darkroom.

[0170]

[0171] HA-DAB synthesis

[0172] To synthesize HA-DAB, HA with a MW of 100 kDa was dissolved in a solvent mixture of ethanol / water (1:1 volume ratio), and then DAB (40 molar ratio of HA repeat units) was dissolved in the HA-containing solution.

[0173] Next, EDC and NHS (2 molar ratios 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 1 N HCl aqueous solution. The reaction was carried out with vigorous stirring at room temperature for 24 h, and the resulting HA-DAB conjugate was dialyzed against 100 mM NaCl solution for 3 days, 25% ethanol solution for 1 day, and DI water for 1 day.

[0174] The purified conjugate solution was lyophilized for 3 days. The effectiveness of HA modification was determined by proton nuclear magnetic resonance (Fig. 11 (AVANCE NEO 500, Bruker, Germany) 1 H NMR) spectrum was analyzed.

[0175]

[0176] Fabrication of nanoplatforms of UCNPs@SiO2-Ce6-HA (referred to as "UCNPs@SiO2-Ce6-HA" or "UCNPs@SiO2-Ce6-HA")

[0177] The prepared HA-DAB was conjugated to the surface of UCNPs@SiO2-Ce6 (10 mg) via 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 h.

[0178] The resulting suspension was dialyzed against deionized water in a dialysis bag with a molecular weight cutoff of 7 kDa for 3 days to remove unreacted molecules. The final lyophilized product was stored at -20°C in the dark for future use.

[0179]

[0180] <Experimental Example>

[0181] Characteristic evaluation

[0182] Sample morphology images were confirmed using an 80 kV transmission electron microscope (TEM, Talos, FEI, Lausanne, Switzerland).

[0183] 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).

[0184] Carbon contamination on the TEM grid was removed using an ion cleaner JIC-410 at 285 V for 5 min (JEOL, Tokyo, Japan).

[0185] Crystallographic analysis was performed using Vesta software (Vesta Software Group, Wallingford, UK).

[0186] Upconversion emission spectra were obtained using a spectrofluorometer (FL-1039, HORIBA Scientific Co., Kyoto, Japan) using an 808 nm infrared diode laser system as the excitation light source. The laser output power density was adjusted using a power meter (843-R, Newport Corp., Irvine, USA).

[0187] Qualitative analysis of functional groups on the nanoparticle surface was performed using a Spectrum Two Fourier Transform Infrared Spectrometer (Perkin Elmer Inc., Waltham, MA, USA).

[0188] The crystal structure of UCNPs@SiO2powder was analyzed using a PANalytical X'Pert Pro X-ray diffractometer.

[0189] UV / vis absorption spectra were measured using a Scinco Mega-800 spectrophotometer (Seoul, Korea). The lanthanide composition of UCNPs 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).

[0190] Particle size distribution analysis was performed using ImageJ software (Rasband, WS, ImageJ, US National Institutes of Health, Bethesda, Maryland, USA).

[0191] Geometric mean diameter d g,p and geometric standard deviation σ g was calculated using the equation shown below:

[0192] (1)

[0193] (2)

[0194]

[0195] Here, N is the total number of samples and d i is the particle size measured from TEM images.

[0196]

[0197] Extracellular detection of reactive oxygen species

[0198] 1 mL of UCNPs@SiO2-Ce6-HA (1 mg / mL) aqueous solution was mixed with 1 mL of DPBF solution dissolved in ethanol in a 4 mL cuvette, and then the sample was exposed to 0.5 W / cm 2 was exposed to an 808 nm laser at a power density of .

[0199] UV-Vis absorption was measured at approximately 410 nm for 10 min (0, 2, 4, 6, 8, and 10 min). The low-temperature effect was confirmed by keeping the cuvette at -80°C for 1 min before laser irradiation.

[0200]

[0201] Cell uptake experiments

[0202] Cellular uptake was assessed using L929 and B16F10 cell lines purchased from the Korean Cell Line Bank (Seoul, Korea). Cells were seeded at 1 x 10 per well. 5 The cells were cultured in 12-well plates at a density of 10 cells / well. The cells were cultured in standard medium containing 10% FBS and 1% antibiotics at 37°C in an environment containing 5% CO2 for 1 day.

[0203] Then, the cells were incubated with a nanoparticle suspension at a concentration of 500 μg / mL for 4 h. The cells were washed twice with PBS solution before fixation with 4% paraformaldehyde.

[0204] Next, cells were stained with DAPI for 10 min before imaging using a confocal laser scanning microscope (ZEISS LSM 800, Oberkochen, Germany). To understand the role of the HA receptors CD44 and LYVE-1 during nanoparticle uptake, competitive uptake inhibition was performed in B16F10 cells by adding free HA (100 kDa, 10 mg / mL, 200 μL) for 30 min.

[0205]

[0206] Live / Dead Cell Assessment

[0207] B16F10 cells were seeded at 0.5х10 per well in a 24-well plate 5The cells were cultured at a density of 10 μg / mL. The cells were cultured in a standard medium containing 10% FBS and 1% antibiotics at 37°C in an atmosphere containing 5% CO2 for 1 day. After culturing the cells for 24 h, the cells were incubated with a nanoparticle suspension at a concentration of 500 μg / mL for 4 h and then irradiated with an 808 nm laser at 0.5 W / cm 2 Cells were irradiated for 10 min at a power density of . Cells were incubated with Live / Dead cell assay solution for 10 min before imaging with a fluorescence microscope (EVOS M5000, Thermo Fisher Scientific, Waltham, MA, USA).

[0208]

[0209] Cell viability analysis by CCK assay

[0210] Cytotoxicity of the samples was assessed by CCK-8 (tetrazolium salt) assay. B16F10 cells were seeded at 1X10 per well. 4 The cells were cultured in 96-well plates at a density of 10 μg / mL. The cells were cultured overnight at 37°C in a 5% CO2 atmosphere in standard medium containing 10% FBS and 1% antibiotics. The cells were then incubated with nanoparticle suspensions at different concentrations (10, 50, 100, 200, and 500 μg / mL) for 24 h.

[0211] Next, the cell medium was replaced, and 10 μL of 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.

[0212]

[0213] Assessment of intracellular singlet oxygen production

[0214] B16F10 cells were seeded at 0.5х10 per well in a 24-well plate 5The cells were cultured at a density of 10 μg / mL. After culturing the cells for 24 h, the cells were incubated with a nanoparticle suspension at a concentration of 500 μg / mL for 4 h and then exposed to an 808 nm laser at 0.5 W / cm 2 Cells were irradiated at a power density of 10 for 10 min. Cells were incubated with the DCFH-DA probe for 30 min before imaging with a fluorescence microscope.

[0215]

[0216] In vitro photodynamic therapy

[0217] B16F10 cells were seeded at 1X10 per well in a 96-well plate 4 The cells were cultured for 24 h at a density of 100 μg / mL. Then, the cells were cultured with nanoparticle suspensions at various concentrations (10, 50, 100, 200, and 500 μg / mL). After 4 h of incubation, the cells were exposed to 0.5 W / cm 2 irradiation, except for the control group. 2 The cells were irradiated with an 808 nm laser at a power density of 10 for 10 min. After laser irradiation, the cells were incubated for 12 h, washed three times with PBS, and incubated with a CCK-8 assay to quantify relative cell viability.

[0218]

[0219] Transdermal delivery of UCNPs@SiO2-Ce6-HA nanoplatform

[0220] The penetration ability of the UCNPs@SiO2-Ce6-HA nanoplatforms across the skin layer was evaluated using freshly excised pig skin obtained from a local slaughterhouse. UCNPs@SiO2-Ce6 and UCNPs@SiO2-Ce6-HA nanoplatforms were dispersed in PBS at a concentration of 500 μg / mL and topically applied to the pig skin surface sectioned into 0.5 × 0.5 cm sections using a freezing microtome (Leica, CM1860, Wetzlar, Germany).

[0221] Transdermal delivery was evaluated using a confocal laser scanning microscope (ZEISS LSM 800, Oberkochen, Germany) at 2, 4, 6, 12, and 24 h. Fluorescence intensity was measured using ImageJ software.

[0222]

[0223] In vivo antitumor effects of cryo-photodynamic therapy (Cryo-PDT)

[0224] Female C57BL / 6NCrlOri mice, 5–6 weeks old, 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. Mice were randomly divided into several groups before treatment.

[0225] To evaluate the antitumor Cryo-PDT effect, mice were injected with 5 Х 10 6 Tumors were induced by subcutaneously injecting B16F10 cells, which are melanoma cells, at a density of 100 μM. B16F10 cells were suspended in DMEM and injected into the right dorsal flank.

[0226] One week after tumor cell inoculation, mice were randomly divided into seven treatment groups (n = 4 each 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).

[0227] At the treatment stage, the tumor volume is on average 70 mm 3 Tumor growth was monitored using the following formula: Tumor volume = A Х 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 locally administered to the tumor surfaces of groups 2, 5, 6, and 7 for 30 min.

[0228] Cryotherapy in 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. Laser groups 4 and 6 received an 808 nm laser at 0.5 W / cm 2 was irradiated for 10 minutes at a power density of 0.5 W / cm. In particular, Cryo-PDT combination treatment in group 7 was performed through 10 consecutive cycles, each cycle consisting of 1 minute Cryo (liquid nitrogen) treatment and 1 minute laser treatment (0.5 W / cm 2 ) was composed of. Each Cryo cycle includes 30-second freeze-thaw cycles.

[0229] The laser setup is illustrated in Figure 17. Laser output 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 tumor volume to initial tumor volume.

[0230]

[0231] Histological analysis of tumor cell death

[0232] After 14 days of treatment, tumor tissues were collected from all treatment groups, washed with PBS, and stored in 4% paraformaldehyde. Tumor tissues were dissected and embedded in paraffin. To determine the degree of tumor cell death, 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 manufacturers' protocols.

[0233]

[0234] Statistical analysis

[0235] Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA) using a one-way ANOVA. Values ​​of *, P < 0.05; **, P < 0.01; and ***, P < 0.001 were considered significant. All data are presented as the mean ± standard deviation (SD) from at least three independent experiments.

[0236]

[0237] Characterization of upconversion nanoparticles

[0238] Figure 4A illustrates a schematic diagram of the synthesis route of the upconversion nanoparticles of the present invention. The upconversion nanoparticles of the present invention can precisely control the sodium fluoride content during synthesis by injecting solution B, a precipitant, into solution A.

[0239] In particular, a dual nitrogen (N2) gas balloon system was applied during synthesis to stabilize the pressure, as a pressure increase after injection due to residual methanol and moisture was inevitable. Furthermore, the effect of post-injection synthesis time on particle size distribution was investigated, and the results are shown in Figure 2.

[0240] Referring to Figure 2, it can be seen that synthesis for 2.5 hours yields an optimal uniform size, which is indicated by a clear size distribution profile and a geometric standard deviation (σ) of 1.046. g ) was confirmed.

[0241] FIG. 4B is a TEM image of the upconversion nanoparticles of the present invention. Referring to FIG. 4B, it can be confirmed that monodisperse upconversion nanoparticles having a particle size of ~32 nm were produced.

[0242] As a result, the above synthetic conditions were shown to form final homogeneous core@shell (NaYF4:Yb,Tm@NaYF4:Nd) nanoparticles.

[0243] To verify the formation of the hexagonal crystal structure, the selected area electron diffraction (SAED) pattern of single NaYF4:Yb,Tm@NaYF4:Nd nanoparticles was analyzed by high-resolution transmission electron microscopy (HR-TEM).

[0244] As shown in Fig. 4C, the interplanar spacings within the ring pattern were measured to be 5.185Å, 2.999Å, 1.960Å, and 1.740Å, which were in good agreement with the planes (100), (110), (210), and (002) of the hexagonal β-NaYF4.

[0245] In addition, the presence of Y, Yb, Tm, and Nd elements was confirmed by mapping the element distribution using HAADF-EDS (high-angle annular dark-field imaging coupled with energy dispersive spectroscopy), and the results are shown in Fig. 5.

[0246] According to ICP-OES (Inductively Coupled Plasma Optical Emission Spectrometry) analysis, the molar ratio of Y:Yb:Tm:Nd in UCNPs was 81.5:9.0:0.1:9.4 (Table 1). All of these results confirmed the successful synthesis of UCNPs of the present invention.

[0247] Element [mol.%]Y, mol.%Yb, mol.%Tm, mol.%Nd, mol.%Theoretical80.39.50.110Experimental81.59.00.19.4

[0248] To investigate the effect of temperature on UCNP emission, the photoluminescence (PL) emission spectra of UCNPs in cyclohexane suspension were obtained for the same sample at room temperature (295 K) and post-thaw conditions under 808 nm laser excitation (0.5 W / cm 2 ) was recorded under the conditions, and the results are shown in Fig. 4D. "Post-thaw" refers to a sample that was stored in a freezer at 193 K for 10 minutes before PL measurement, and then thawed at 295 K. In the present invention, it was confirmed that the area of ​​the total PL emission peak increased by 2.45 times under the post-thaw condition, which can be interpreted as the effect of exposure to freezing.

[0249] Exposure to low temperature environments limits phonon-induced cross-relaxation and excitation states (e.g. 1 I6, 1 D2 and 1 The density of G4) was maximized, increasing the radiation emission.

[0250] Figure 6 is a schematic diagram of the upconversion process in the upconversion nanoparticles of the present invention. In Figure 6, Yb 3+ is 808 nm excitation Nd 3+ By transmitting the photons harvested from the photosensitizer to Tm 3+ It acts as an energy mediator to photoactivate emitter ions. Under NIR laser irradiation, Nd 3+ The ion harvests 808 nm photons and converts them to Yb by a cascade energy transfer mechanism as shown in Fig. 6. 3+ ion( 2 F 5 / 2 → 2 F 7 / 2 ) through which energy is transferred here to Tm 3+ ion( 3H6) was transferred to the ground state.

[0251] As a result, Tm 3+ The emission 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 at H6.

[0252] After thawing, the blue upconversion emission of UCNPs was readily observed with the naked eye under on- and off-state incandescent light conditions (inset image in Figure 4D). Therefore, the clear advantage of cryogenically induced modulation of upconversion brightness was confirmed for developing a synergistic treatment strategy combining cryotherapy and photodynamic therapy.

[0253]

[0254] Confirmation of UCNP@SiO2 characteristics

[0255] Figure 3 is a low-resolution TEM image (scale bar: 1 μm) of UCNP@SiO2 nanoparticles to verify uniform coating without severe aggregation. The synthesis was performed under continuous sonication to ensure the single encapsulation of UCNPs, which was intended to isolate the UCNPs before encapsulating them in a silica layer to prevent unwanted aggregation.

[0256] Successful encapsulation of UCNPs@SiO2 nanoparticles in a porous silica layer was confirmed by TEM, as shown in Fig. 4E. Referring to Fig. 7, the geometric mean diameter of UCNPs@SiO2 nanoparticles was 76.214 nm, indicating that the average thickness of the silica layer surrounding the UCNPs was approximately 22 nm.

[0257] Figure 4F shows the fast Fourier transform (FFT) pattern of the HR-TEM image of UCNPs@SiO2 nanoparticles, through which information on the local diffraction pattern was obtained, confirming that the hexagonal crystal structure of UCNPs was maintained without damage.

[0258] Referring to Fig. 4F, the interlattice spacings were measured to be 5.112 Å (yellow dashed circle) and 5.192 Å (green dashed circle), respectively, which are consistent with the hexagonal lattice parameter (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 characteristic broad peaks and sharp peaks of amorphous silica, which are consistent with 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 was maintained even after the silica layer was formed under ultrasonic destruction synthesis conditions.

[0259] Figure 4H shows the HAADF-STEM image and HAADF-EDS elemental mapping of UCNPs@SiO2 nanoparticles, which further verified the uniform distribution of representative elements such as Y, Yb, Tm, and Nd in the core structure and Si element in the outermost shell, confirming the successful fabrication of UCNPs@SiO2. The PL analysis results showed that the integrated PL intensity decreased by 22.9% after silica shell encapsulation, which could be attributed to the absorption by the outer silica layer (Figure 8).

[0260]

[0261] Confirmation of nanoplatform characteristics

[0262] Figure 9A is a schematic diagram for the fabrication of the UCNPs@SiO2-Ce6-HA nanoplatform. Prior to the synthesis of Ce6 and HA, UCNPs@SiO2 was surface-modified with 3-aminopropyltriethoxysilane (APTES) to introduce amine groups. This was confirmed by a change in zeta potential from negative (-25.8 ± 0.84 mV) to positive (38.75 ± 1.04 mV). UCNPs@SiO2-NH2 was then conjugated with Ce6 via an EDC / NHS chemical reaction.

[0263] The loading efficiency of Ce6 in the UCNPs@SiO2-Ce6 composite was estimated to be 29.2 wt.%, as determined by the calibration curve of the standard Ce6 solution (Fig. 10). The zeta potential changed to a negative value of -29.84 ± 0.17 mV, reflecting the abundant carboxyl moieties on the surface of UCNPs@SiO2-Ce6. Subsequently, UCNPs@SiO2-Ce6 were conjugated with HA by the reaction of the carboxyl groups of UCNPs@SiO2-Ce6 with the amine groups of the HA-diaminobutane (DAB) conjugate.

[0264] 1 H NMR analysis showed that the characteristic peaks observed at δ1.9 (HA) and δ1.6 (DAB) portions arising from the methyl moieties of HA and DAB, with a DAB ratio of 32.25% (Fig. 11). TEM images of the UCNPs@SiO2-Ce6 and UCNPs@SiO2-Ce6-HA nanoplatforms confirmed the successful surface modification of UCNPs@SiO2 (Figs. 9B and 9C).

[0265] Due to the HA coating, the zeta potential was slightly shifted to a negative value of -33.16 ± 0.03 mV, which provided stable colloidal behavior in a physiological environment. 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 had a narrow size distribution (PDI: 0.2448, nearly monodisperse).

[0266] The evolution of molecular bonding during the fabrication of UCNP@SiO2-Ce6-HA nanoplatforms was evaluated using FT-IR spectroscopy (Fig. 9D). 1065 cm -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.

[0267] Ce6 loading is 1538 cm -1 (COO- Stretching) and 1633 cm -1 (C=N stretching) was confirmed as a new peak. The surface modification of UCNPs@SiO2-Ce6 with HA was observed at 3291 cm -1 (OH Stretching), 2882 cm -1 (CH stretching), 1394 cm -1 and 1376 cm -1 (CH bend) and 1045 cm -1 (CO stretching) generated a peak in the absorption band.

[0268] The normalized absorbance profile showed a signal at 410 nm (Figure 9E), which corresponded to the contribution of Ce6, confirming the successful fabrication of the UCNPs@SiO2-Ce6-HA nanoplatform.

[0269] To better understand the photochemical phenomena occurring under 808 nm NIR laser irradiation, singlet oxygen ( 1 O2) production was quantified by detecting it using a probe molecule. The UCNPs of NaYF4:Yb,Tm@NaYF4:Nd converted several 808 nm NIR photon energies into high-energy UV photons, which were then quantified by the Ce6 photosensitizer. 3 O2 cytotoxicity 1 O2 could trigger a photochemical conversion.

[0270] In the present invention, a 1,3-diphenyl isobenzofuran (DPBF) sensor is used to convert colorless 1,3-diphenylisobenzoylbenzene. 1 O2 was captured. 1 O2 production can be quantitatively estimated from the gradual decrease in characteristic absorption intensity around 410 nm in the UV-vis spectrum.

[0271] To investigate the temperature-responsive photo-singlet oxygen conversion, the DPBF decomposition rate was evaluated for 10 min under cryogenic (193 K) and room temperature (295 K) conditions, as shown in Figures 9F and 12. Surprisingly, at 808 nm (0.5 W / cm 2 ) under NIR laser irradiation, the UCNPs@SiO2-Ce6-HA nanoplatform was synthesized under cryogenic conditions (slope 0.0555, R 2 =0.997) exhibited significantly higher degradation kinetics. Relatively moderate photooxidation of DPBF was observed at room temperature (slope 0.0176, R 2 = 0.997).

[0272] As a result, when irradiated with a near-infrared laser for 10 minutes under cryogenic conditions, the UCNPs@SiO2-Ce6-HA nanoplatform 1 It was confirmed that O2 production was improved by 3.15 times.

[0273]

[0274] 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. No noticeable toxicity was observed in mouse fibroblast L929 cells and B16F10 melanoma cells due to the UCNPs@SiO2-Ce6-HA nanoparticles (Fig. 13).

[0275] To visualize the cellular uptake, fluorescence imaging of UCNPs@SiO2-Ce6-HA was performed. Referring to Fig. 14A, confocal laser scanning microscopy (CLSM) visualized the penetration of the UCNPs@SiO2-Ce6-HA nanoplatform into L929 and B16F10 cells. UCNPs@SiO2-Ce6-HA showed a 1.74-fold increase in red fluorescence signal intensity in B16F10 cells compared to L929 cells (Fig. 14B). Using these characteristics, it was confirmed that UCNPs@SiO2-Ce6-HA selectively accumulated only in tumor cells while minimizing damage to normal cells.

[0276] Confocal laser scanning microscopy (CLSM) clearly visualized the effective penetration of UCNPs@SiO2-Ce6-HA nanoplatform into B16F10 melanoma cells via HA receptor-mediated endocytosis (Fig. 15A).

[0277] HA receptors such as cluster determinant 44 (CD44) and lymphatic endothelial hyaluronan receptor-1 (LYVE-1) were inactivated by pre-incubating B16F10 cells with excessive amounts of HA, which showed a significant decrease in cellular uptake of the UCNP@SiO2-Ce6-HA nanoplatform due to competitive binding of HA.

[0278] These results demonstrated that the UCNPs@SiO2-Ce6-HA nanoplatform was taken up by HA receptor-mediated endocytosis.

[0279] Referring to Figure 15A, in the absence of HA, the red fluorescence intensity of Ce6 around the nucleus dramatically increased, reflecting the efficient cellular uptake of the UCNPs@SiO2-Ce6-HA nanoplatform via HA receptor-mediated endocytosis. These results confirmed the role of HA in the tumor-targeted accumulation of UCNPs@SiO2-Ce6-HA.

[0280] The antitumor therapeutic efficacy of the UCNPs@SiO2-Ce6-HA nanoplatform was evaluated by in vitro photodetector-beam-detection (PDT). Intracellular singlet oxygen generation was visualized by singlet oxygen-induced conversion of DCFH-DA to oxidized DCF, which exhibits green fluorescence.

[0281] Figure 15B shows fluorescence microscopy images of the control, laser, and UCNPs@SiO2-Ce6-HA treatment groups, where green fluorescence was indistinct, confirming no or minimal singlet oxygen production. In contrast, clear green fluorescence was observed in B16F10 cells cultured with UCNPs@SiO2-Ce6-HA and then irradiated 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.

[0282] 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 with UCNPs@SiO2-Ce6-HA nanoplatforms at various concentrations from 0 to 500 μg / mL for 24 h. Afterwards, the cells were irradiated with 0.5 W / cm 2 The 808 nm NIR laser was irradiated for 10 minutes at a power density of .

[0283] Referring to Figure 15C, the viability of B16F10 cells decreased to 22.3% at a concentration of 500 μg / mL upon 808 nm NIR irradiation. To further verify the previously conducted experimental results, the in vitro antitumor therapeutic effect was visualized through simultaneous staining analysis of live and dead cells using Calcein AM and propidium iodide (PI).

[0284] In Fig. 15D, no significant toxicity was observed in the control, laser, and baseline treatments using UCNPs@SiO2-Ce6-HA nanoplatform, whereas treatment using UCNPs@SiO2-Ce6-HA nanoplatform and 808 nm laser (Laser + UCNPs@SiO2-Ce6-HA) showed almost no green fluorescence (live cells) and strong red fluorescence (dead cells). These results confirmed that the UCNPs@SiO2-Ce6-HA nanoplatform exhibited high biocompatibility and near-infrared-mediated anticancer efficacy.

[0285] 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.

[0286] HA is one of the essential biological components of the epidermis and dermis of human skin and is known to promote transdermal drug delivery deep into the dermis.

[0287] Figure 15E shows confocal microscopy scanning images and depth profiles of cryosectioned porcine skin surfaces after topical administration of UCNPs@SiO2-Ce6 and UCNPs@SiO2-Ce6-HA nanoplatform solutions in PBS in vitro. The extent of skin penetration was monitored for 24 h using the red fluorescence signal of the Ce6 photosensitizer in the skin. Figures 15E and 16 show a significant difference in skin penetration between UCNPs@SiO2-Ce6-HA nanoplatforms and UCNPs@SiO2-Ce6 nanoparticles. The HA-coated nanoparticles were confirmed to be evenly distributed in the deep layers of the skin with bright red fluorescence. The images in Figure 16 were 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).

[0288] The intensity gradually increased over time, reflecting that the UCNPs@SiO2-Ce6-HA nanoplatform exhibited effective transdermal delivery through the stratum corneum and basal porcine skin layers. These results were also confirmed by the depth profile analysis shown in Figure 15F.

[0289] More specifically, the integrated red fluorescence intensity area in the epidermal layer increased 3.2-fold between 2 and 24 h. As expected, the diffusion of UCNPs@SiO2-Ce6 was impeded by the skin barrier and mainly accumulated in the stratum corneum. These results were well consistent with other reports, demonstrating the excellent potential of the UCNPs@SiO2-Ce6-HA nanoplatform for effective transdermal delivery into 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.

[0290] The in vivo antitumor effect of the UCNPs@SiO2-Ce6-HA nanoplatform was further confirmed in C57BL / 6 mice bearing B16F10 tumors. To maximize the PDT efficacy of the UCNPs@SiO2-Ce6-HA nanoplatform, cryotherapy was used for melanoma tumors. The B16F10 tumor-bearing mice were randomly divided into seven groups as follows: (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), (7) Cryo + Laser + UCNPs@SiO2-Ce6-HA (Cryo-PDT).

[0291] The tumor sites of groups 2, 5, 6, and 7 were locally treated with UCNPs@SiO2-Ce6-HA solution (500 μg / mL, 50 μL) dispersed in PBS for 30 min. In cryogroups 3 and 5, the tumor sites were treated with liquid nitrogen for 10 min. Laser groups 4 and 6 were treated with an 808 nm NIR laser (0.5 W / cm 2 , 10 minutes) was investigated.

[0292] Figure 17 is a schematic diagram of the laser setup. The laser output density was adjusted using a power meter (843-R, Newport, USA). In Group 7, local administration of the UCNPs@SiO2-Ce6-HA solution (30 min) was followed by 1-min Cryo and 1-min Laser cycle treatment (total 20 min).

[0293] Figure 18 shows the thermal mapping obtained from a single Cryo / Laser treatment cycle, and is the result of thermal images captured during a Cryo-PDT cycle comprising 1 minute Cryo and 1 minute Laser treatment. Each Cryo cycle included 30-second freeze-thaw cycles. The thermal images were captured using a FLIR infrared camera, with low-temperature sensitivity limited to -20°C.

[0294] Figure 19A shows the temperature fluctuation curve. The 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 min.

[0295] Confocal microscopy scanning images showed that the UCNPs@SiO2-Ce6-HA nanoplatform exhibited clear red fluorescence in the deep region, including the skin and the tumor tissue beneath the skin, in the entire observed cancer tissue (Fig. 19B). In contrast, UCNPs@SiO2-Ce6 exhibited strong fluorescence in the upper skin surface region of the tumor tissue, indicating poor penetration into the tumor beneath the skin.

[0296] After treatment, mice were sacrificed and digital photographs of the collected tumors were taken, as shown in Figure 19C, to clearly visualize the antitumor effect of the treatment group.

[0297] Tumor volumes and body weights of mice were measured for 14 days. Figure 19D shows that the body weights of mice did not decrease significantly during the treatment period, indicating that there was no significant toxicity caused by UCNPs@SiO2-Ce6-HA, laser, and Cryo treatments. The administered nanoparticle doses appeared to induce negligible toxicity in vivo. As shown in Figure 19E, the UCNPs@SiO2-Ce6-HA and laser groups showed similar tumor inhibition rates to the control group. A slight tumor growth delay was observed in the Cryo and Cryo + UCNPs@SiO2-Ce6-HA groups, where the tumor growth inhibition rates were similar at approximately 17%.

[0298] Notably, statistically significant tumor growth inhibition was observed in mice treated in the PDT group. 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%. The successful Cryo-PDT treatment may be attributed to the sequential Cryo and Laser treatment cycles that maintain cooling of the tumor site for effective upconversion of UCNPs.

[0299] Finally, tumor sections were stained with hematoxylin and eosin (H&E) and subjected 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.

[0300] In particular, the tumor histological structure and cell viability did not change significantly in the control, UCNPs@SiO2-Ce6-HA, and laser groups. Slight morphological changes and cell death were observed in the Cryo and Cryo + UCNPs@SiO2-Ce6-HA groups. Consistent with tumor growth, the tumor treated with Cryo-PDT treatment group (Cryo + Laser + UCNPs@SiO2-Ce6-HA) showed the most severe tissue damage and prominent cell death and necrosis in the H&E staining images.

[0301] The TUNEL assay also supported the antitumor effect, with cell death exhibiting a strong green fluorescent signal in key areas compared to PDT treatment alone without the Cryo effect. In summary, the present invention successfully demonstrated the remarkable synergistic therapeutic efficacy of the Cry-PDT treatment against melanoma.

[0302]

[0303] While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the exemplary embodiments disclosed herein, and that various modifications may be made by those skilled in the art within the scope of the technical spirit of the invention. Furthermore, even if the operational effects of the configurations of the invention have not been explicitly described and explained while describing the exemplary embodiments of the invention, it is also to be understood that the effects predictable by the configurations should be acknowledged.

Claims

1. NaYF 4 :Core consisting of Yb, Tm; and NaYF surrounding the above core 4 :A nanoplatform comprising core-shell structured upconversion nanoparticles including a shell made of :Nd.

2. In paragraph 1, The above nanoplatform is the NaYF 4 :SiO surrounding a shell made of Nd 2 A nanoplatform further comprising a porous shell comprising:

3. In paragraph 1, The above-mentioned upconversion nanoparticles are nanoplatforms manufactured through a single-step synthesis.

4. In paragraph 2, The porous shell may be comprised of chlorin e6, zinc phthalocyanine (ZnPC), aminolevulinic acid (ALA), methyl aminolevulinate, temoporfin, phthalocyanine, protophorap IX (PpIX), Allumera™, Cevira™, Hexvix™, porfimer sodium, verteporfin, δ-aminolevulinic acid or 5-aminolevulinic acid, temoporfin, methyl aminolevulinate, hexaminolevulinate hydrochloride, talaporfin, motexafin lutetium, 2-(1-hexyloxyethyl)-2-devinyl A nanoplatform loaded with at least one photosensitizer selected from the group consisting of 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a, Photosens™, rostaporfin, BF-200 ALA, tetraphenyl chlorin disulfonate, and azadipyrromethene.

5. In paragraph 2, The above porous shell is a nanoplatform to which hyaluronic acid is bound.

6. In paragraph 1, A nanoplatform, wherein the size of the above upward conversion nanoparticles is 10 to 100 nm.

7. In paragraph 2, A nanoplatform, wherein the thickness of the porous shell is 1 to 50 nm.

8. A pharmaceutical composition for preventing or treating cancer, comprising a nanoplatform according to any one of claims 1 to 6.

9. In paragraph 7, A pharmaceutical composition for preventing or treating cancer, wherein the cancer is at least one 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, gastric cancer, esophageal cancer, anal cancer, biliary tract cancer, 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.

10. In paragraph 7, A pharmaceutical composition for the prevention or treatment of cancer, wherein the treatment is selected from the group consisting of photodynamic therapy, cryotherapy and combinations thereof.

11. A food composition for preventing or improving cancer, comprising a nanoplatform according to any one of claims 1 to 6.

12. In Article 10, A food composition for preventing or improving cancer, wherein the cancer is at least one 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, gastric cancer, esophageal cancer, anal cancer, biliary tract cancer, 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.

13. A method for treating cancer, comprising a step of administering a therapeutically effective amount of a nanoplatform according to Article 1 to a subject.

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