Mesoporous ceria nanoparticles for prevention, treatment, or alleviation of ophthalmic diseases

Abstract

Disclosed herein are mesoporous ceria nanoparticles for the prevention, treatment, or alleviation of ophthalmic diseases. In one aspect, having mesoporous structure with high surface area and pore size and mimicking the catalytic properties of catalase and superoxide dismutase (SOD), the mesoporous ceria nanoparticles of the present invention have excellent ability to scavenge reactive oxygen species, exhibit anti-inflammatory effects, demonstrate biocompatibility without causing acute side effects or ocular toxicity in the retina, and are recognized to provide protective effects against cellular damage and protect damaged retinal pigment epithelial (RPE) cells and photoreceptors under high oxidative stress. Therefore, the nanoparticles can be utilized for the prevention, treatment, or alleviation of macular degeneration and various oxidative stress-mediated ophthalmic diseases.

Description

Mesoporous ceria nanoparticles for preventing, treating, or improving ophthalmic diseases

[0001] Disclosed herein are mesoporous ceria nanoparticles for preventing, treating or improving ophthalmic diseases.

[0002]

[0003] Cross-reference to related applications

[0004] This application claims priority to Republic of Korea Patent Application No. 10-2024-0017987, filed February 6, 2024, the entire contents of which are incorporated herein by reference.

[0005]

[0006] Meanwhile, this application was supported by the following national development project.

[0007] [National Research and Development Project Supporting This Invention]

[0008] [Project ID] 1465040446

[0009] [Assignment Number] HI23C1514

[0010] [Ministry Name] Ministry of Health and Welfare

[0011] [Name of Project Management (Specialist) Agency] Korea Health Industry Development Institute

[0012] [Research Project Name] K-Medi Convergence Talent Development Support Project

[0013] [Research Project Title] Development of a Permanent Nanocatalytic Intraocular Lens for the Treatment of Age-Related Macular Degeneration

[0014] [Name of Project Performing Organization] Seoul National University Hospital

[0015] Research Period: April 1, 2023 - December 31, 2024

[0016]

[0017] [National Research and Development Project Supporting This Invention]

[0018] [Project ID] 1711189569

[0019] [Assignment Number] 2022R1A2B5B02002097

[0020] [Ministry Name] Ministry of Science and ICT

[0021] [Name of Project Management (Specialist) Institution] National Research Foundation of Korea

[0022] [Research Project Name] Individual Basic Research (Ministry of Science and ICT)

[0023] [Research Project Title] Development of a Nanoparticle-Based Multiple Sclerosis Treatment Vaccine that Induces Enhanced Antigen-Specific Immune Tolerance

[0024] [Name of the project performing organization] Sungkyunkwan University

[0025] [Research Period] March 1, 2023 - February 29, 2024

[0026]

[0027] Age-related macular degeneration (AMD) is an acquired retinal disease characterized by progressive vision loss due to degenerative changes in the photoreceptors and retinal pigment epithelium of the macula. It is the leading cause of vision loss in people over 65 years of age, and it is reported that over 8 million patients are affected in the United States alone. AMD can eventually cause vision loss and blindness, which significantly reduces the quality of life in old age, frequently causes depression, and can cause enormous economic losses due to treatment and purchase of vision aids. Therefore, the development of a treatment is urgent. However, at present, only symptomatic treatments such as intravitreal injections of ranibizumab, aflibercept, bevacizumab, brolucizumab, and VABYSMO (faricimab) are used clinically, rather than radical treatments.

[0028] Although various factors play a role in the onset and worsening mechanism of age-related macular degeneration, the role of excessive reactive oxygen species and increased inflammation in the eye have recently been receiving attention. First, when reactive oxygen species are excessively generated, it causes lipid peroxidation of cell membranes, oxidative changes in proteins, and cell death and mutations due to oxidative damage to DNA. This action is known to play an important role in the development of various diseases in the field of ophthalmology, such as cataracts, uveitis, retinopathy of prematurity, age-related macular degeneration, primary open-angle glaucoma, corneal aging, keratitis, dry eye, keratoconus, bullous keratopathy, and Fuchs' endothelial dystrophy. In the retina affected by age-related macular degeneration, extracellular deposits called drusen are observed. These are known to be adducts of advanced glycation end-products (AGEs) and carboxyethyl-pyrrole, which are caused by oxidative damage to fatty acids located at the tips of photoreceptors. In addition, mice that have knocked out Nuclear factor erythroid 2-related factor 2 (NRF2), a transcription factor responsible for the antioxidant defense mechanism of the retina, develop drusen-like deposits, lipofuscin accumulation, choroidal neovascularization, and subretinal deposition of inflammatory proteins, which are major features of human age-related macular degeneration. Thus, excessive reactive oxygen species are significantly involved in the pathogenesis of age-related macular degeneration, and effective removal of excessive reactive oxygen species in the eye and retina may be a new therapeutic alternative for age-related macular degeneration.

[0029] The second factor contributing to the onset and worsening of age-related macular degeneration is increased intraocular inflammation. If chronic inflammation persists within the eye, it can cause degenerative damage to the macular structure. Non-steroidal anti-inflammatory drugs (NSAIDs) can exert therapeutic effects by controlling this inflammation. Indomethacin (IDM), a NSAID, can be used topically to reduce intraocular prostaglandin E2. Furthermore, it has been shown that anatomical and visual outcomes improve when indomethacin is used concurrently with ranibizumab, a treatment for age-related macular degeneration. This suggests that NSAIDs may be helpful in the treatment of age-related macular degeneration.

[0030] Wet AMD, a type of age-related macular degeneration, accounts for 10% of all macular degeneration patients. It is caused by abnormal choroidal neovascularization in the macula, which can cause exudates and hemorrhages in the macula, leading to severe central vision loss and irreversible vision loss. Current treatments for wet AMD include photodynamic therapy and intravitreal anti-vascular endothelial growth factor (anti-VEGF) injections. However, in the drug treatment of age-related macular degeneration, most of the non-steroidal anti-inflammatory drugs mentioned above have very low solubility in water, which significantly reduces bioavailability and reduces the efficacy of the drug. In addition, anti-VEGF intravitreal antibody injection is a symptomatic treatment for symptom control rather than a radical treatment aimed at complete cure, and because it has a short half-life, frequent intravitreal injections are required to maintain the efficacy, which reduces the convenience of administration for patients. In addition, the non-response rate to anti-VEGF is 35-50%, and many cases do not respond to treatment. Therefore, new treatments including the development of effective drug delivery systems are needed.

[0031]

[0032] The purpose of the present invention is to provide mesoporous ceria nanoparticles for preventing, treating or improving ophthalmic diseases.

[0033] Another object of the present invention is to provide a composition for preventing, treating or improving ophthalmic diseases comprising the mesoporous ceria nanoparticles.

[0034] Another object of the present invention is to provide a method for producing the mesoporous ceria nanoparticles.

[0035]

[0036] To achieve the above purpose, the present invention provides, in one aspect, mesoporous ceria nanoparticles containing an anti-inflammatory agent, an antioxidant agent, and / or an ophthalmic disease treatment agent.

[0037] In another aspect, the present invention provides a composition for preventing, treating or improving ophthalmic diseases caused by oxidative stress, comprising the mesoporous ceria nanoparticles.

[0038] In another aspect, the present invention provides a method for producing the mesoporous ceria nanoparticles, comprising the following steps.

[0039] (a) a step of mixing cerium nitrate hexahydrate, imidazole and 1,1'-Carbonyldiimidazole (CDI) in a non-aqueous solution; and

[0040] (b) A step of obtaining mesoporous ceria nanoparticles from the above mixture.

[0041] The above-described problem-solving methods are merely exemplary and should not be construed as limiting the scope of the present application.

[0042]

[0043] In one aspect, the mesoporous ceria nanoparticles of the present invention have a mesoporous structure with a high surface area and pore size, and have excellent reactive oxygen species scavenging ability by mimicking the catalytic properties of catalase and SOD, exhibit an anti-inflammatory effect, do not exhibit acute side effects or ocular toxicity in the retina, exhibit biocompatibility, and have been recognized as having a cell protection effect and a cell damage alleviation effect under high oxidative stress, and have been recognized as having a protective effect on damaged retinal pigment epithelial cells (RPE) and photoreceptors, and therefore can be utilized for the prevention, treatment, or symptom relief of macular degeneration and various oxidative stress-mediated ophthalmic diseases. The mesoporous ceria nanoparticles can be applied not only to the prevention of age-related macular degeneration and ocular tissue protection, but also to the treatment of various ophthalmic diseases that may be caused by an increase in reactive oxygen species in the eye, such as uveitis, open-angle glaucoma, and Fuchs corneal endothelial dystrophy.

[0044] In another aspect, the mesoporous ceria nanoparticles of the present invention can effectively enhance the anti-inflammatory effect and therapeutic effect by loading an anti-inflammatory drug (e.g., NSAID).

[0045] In another aspect, the mesoporous ceria nanoparticles of the present invention can effectively enhance the effect of preventing, treating, or alleviating symptoms of ophthalmic diseases by loading an antioxidant or an ophthalmic disease treatment agent (e.g., anti-vascular endothelial growth factor (anti-VEGF)), and can be applied to the development and clinical use of highly efficient therapeutic mesoporous ceria nanoparticles capable of simultaneously treating various ophthalmic diseases with various combinations of treatment agents.

[0046] In another aspect, the mesoporous ceria nanoparticles of the present invention can be applied to sustained drug release by allowing the loaded drug to be continuously released for a long period of time.

[0047] In another aspect, the mesoporous ceria nanoparticles of the present invention can be recognized for their versatility by simultaneously delivering the pharmacological effects of various drugs and the effect of removing reactive oxygen species by carrying various drugs such as antibiotics, anticancer drugs, and steroids.

[0048] In another aspect, the present invention can be applied to research on biomaterials based on novel drug-nanocatalyst composites that effectively control oxidative stress in vivo, and to rapid clinical applications. Furthermore, the present invention has potential applications in diverse fields, including research on biomaterials based on drug-nanocatalyst composites that effectively control harmful reactive oxygen species, and applications in treating other local and systemic diseases caused or exacerbated by harmful reactive oxygen species.

[0049] In another aspect, the method for producing mesoporous ceria nanoparticles of the present invention can produce mesoporous ceria nanoparticles without special equipment or additional heating by synthesizing mesoporous ceria nanoparticles using CDI and imidazole in a non-aqueous solution (e.g., acetone).

[0050]

[0051] Figure 1 is a schematic diagram showing a method for treating age-related macular degeneration (AMD) through intravitreal administration of mesoporous cerium oxide (ceria) nanoparticles (mCeNP).

[0052] Figure 2 is a flow chart for manufacturing drug-loaded mesoporous ceria nanoparticles.

[0053] Figure 3 is a scanning electron microscope image of synthesized mesoporous ceria nanoparticles with a size of approximately 120 nm.

[0054] Figure 4 is a transmission electron microscope image of synthesized mesoporous ceria nanoparticles with a size of approximately 120 nm.

[0055] Figure 5 shows the hydrodynamic size and surface charge-related zeta potential data of mesoporous ceria nanoparticles.

[0056] Figure 6 shows the results of N2 adsorption analysis of mesoporous ceria nanoparticles (left) and the results of Brunauer-Emmett-Teller (BET) surface area analysis and Barrett-Joyner-Halenda (BJH) pore size and volume analysis based on the results (right).

[0057] Figure 7 shows the results of a hydrogen peroxide / peroxidase assay (left, catalase activity measurement) and a superoxide dismutase (SOD) determination assay (right, SOD activity measurement) performed to confirm the reactive oxygen species removal ability of mesoporous ceria nanoparticles.

[0058] Figure 8 shows the results of cytotoxicity tests performed using human retinal pigment epithelial cells (hRPEs) (left) and murine macrophage cells (Raw264.7) (right) to evaluate the biocompatibility of the particles. *P < 0.05

[0059] Figure 9 shows the cell viability of human retinal pigment epithelial cells cultured for 24 hours in a medium containing 600 μM hydrogen peroxide, an environment with increased reactive oxygen species that exhibit cytotoxicity. *P < 0.05

[0060] Figure 10 shows the results of a drug loading test using indomethacin (IDM) into mesoporous ceria nanoparticles (left) and the results of a drug release profile (right).

[0061] Figure 11 shows the results of a lactate dehydrogenase (LDH) cytotoxicity assay of indomethacin-loaded ceria nanoparticles tested using murine macrophage cells (Raw264.7).

[0062] Figure 12 shows the results of ELISA for interleukin-6 (IL-6), an inflammatory cytokine, measured after administering each particle (mCeNPs: mesoporous ceria nanoparticles, IDM-mCeNPs: indomethacin-loaded mesoporous ceria nanoparticles) to Raw264.7 cells, culturing them for 6 hours, and then treating them with lipopolysaccharide (LPS) to induce inflammation. The cells were then cultured for 24 hours. *P < 0.05

[0063] Figure 13 shows that intraperitoneal administration of sodium iodate (NaIO3), a strong oxidizing agent, selectively causes cell damage to retinal pigment epithelial cells by reactive oxygen species, thereby enabling the creation of an animal model of age-related macular degeneration. It was also confirmed that the outer nuclear layer (ONL, ▶ section) was partially restored when mesoporous ceria nanoparticles were administered.

[0064]

[0065] Hereinafter, the present invention will be described in detail.

[0066]

[0067] Recently, the importance of 'nanocatalytic medicine', which utilizes medical treatment with nanomaterials that efficiently remove reactive oxygen species, has been greatly emphasized, and among them, cerium oxide (ceria) nanoparticles are in the spotlight in the field of reactive oxygen species removal. In the case of existing drug delivery systems using nanoparticles, the drug delivery vehicle itself is almost always unable to perform any function other than drug loading. In this case, in order to simultaneously provide drug loading and reactive oxygen species removal capabilities to a single nanoparticle, nanoparticles with reactive oxygen species removal capabilities must be additionally re-loaded after drug loading on a single drug delivery vehicle. This can reduce the efficiency in terms of drug and particle loading, and the manufacturing process can also become complicated.

[0068] Accordingly, the present invention has completed the present invention by developing drug-loaded mesoporous ceria nanoparticles that are capable of effectively delivering to tissues and releasing in a sustained manner by imparting mesoporous properties known to exhibit high catalytic power and excellent drug-loading capacity to ceria nanoparticles, and then loading drugs exhibiting anti-inflammatory effects, anti-VEGF drug formulations, etc., onto the mesoporous ceria nanoparticles. The drug-loaded mesoporous ceria nanoparticles have the ability to efficiently remove reactive oxygen species themselves.

[0069] The present invention relates, in one aspect, to mesoporous ceria nanoparticles comprising anti-inflammatory, antioxidant and / or ophthalmic disease treatment agents.

[0070] In an exemplary embodiment, the mesoporous ceria nanoparticles may be mesoporous ceria nanoparticles having reactive oxygen species scavenging ability; and anti-inflammatory activity, antioxidant activity, or pharmacological activity.

[0071] In an exemplary embodiment, the anti-inflammatory agent may be, but is not limited to, a non-steroidal anti-inflammatory drug (NSAID).

[0072] In one exemplary embodiment, the antioxidant and / or ophthalmic agent may be one or more selected from the group consisting of, but not limited to, tetramethylpyrazine, resveratrol, ferulic acid, ethyl ferulate (an analog of ferulic acid), and naringenin, Met 12 and polypeptides consisting of elastin-like polypeptide and αB crystallin, natural flavonoids including luteolin, quercetin, vitamin C, vitamin E, tannic acid, Epigallocatechin gallate (EGCG), and combinations thereof.

[0073] In an exemplary embodiment, the ophthalmic treatment agent may be one or more anti-vascular endothelial growth factor (anti-VEGF) agents selected from the group consisting of, but not limited to, ranibizumab, aflibercept, bevacizumab, brolucizumab, VABYSMO (faricimab), and combinations thereof.

[0074] In an exemplary embodiment, the diameter of the ceria nanoparticles may be 40 nm to 200 nm, and specifically, may be 40 nm or more, 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, 80 nm or more, 100 nm or more, or 110 nm or more, and 200 nm or less, 190 nm or less, 180 nm or less, 170 nm or less, 160 nm or less, 150 nm or less, or 140 nm or less. In the diameter range of the ceria nanoparticles, side effects such as cytotoxicity that may be caused when injected into the eye (ocular) can be minimized.

[0075] In an exemplary embodiment, the surface area of ​​the ceria nanoparticles is 80 m 2 g -1 200 m inland 2 g -1 It can be 80 m specifically 2 g -1 Ideal, 85 m 2 g -1 Above, 90 m 2 g -1 or more than 95 m 2 g -1 Ideally, 200 m 2 g -1 Below, 190 m 2 g -1 Below, 180 m 2 g -1 Below, 170 m 2 g -1 Below, 160 m 2 g -1 Below, 150 m 2 g -1 Below, 140 m 2 g -1 Below, 130 m 2 g -1 Below, 120 m 2 g -1 or less than 110 m 2 g -1 The above-described ceria nanoparticles may be, but are not limited to, capable of carrying significant amounts of anti-inflammatory, antioxidant, and / or ophthalmic treatment agents within the surface area range, and may exhibit significant anti-inflammatory, antioxidant, and / or therapeutic effects.

[0076] In an exemplary embodiment, the pore size (diameter) of the ceria nanoparticles may be from 2 nm to 50 nm, preferably from 2 nm to 30 nm, and more preferably from 2 nm to 10 nm, but is not limited thereto. Within the pore size (diameter) range of the ceria nanoparticles, a significant amount of an anti-inflammatory agent, an antioxidant agent, and / or an ophthalmic disease treatment agent may be loaded, and the anti-inflammatory, antioxidant, and / or therapeutic effects may be significantly exhibited.

[0077] In an exemplary embodiment, the pore volume of the ceria nanoparticles is 0.02 cm 3 g -1 2 cm inside 3 g -1 It can be 0.02 cm, specifically 3 g -1 Above, 0.03 cm 3 g -1 Above, 0.04 cm 3 g -1 Above, 0.05 cm 3 g -1 Above, 0.06 cm 3 g -1 or more than 0.07 cm 3 g -1 Ideally, 2 cm 3 g -1 Below, 1.9 cm 3 g -1 Below, 1.8 cm 3 g -1 Below, 1.7 cm 3 g -1 Below, 1.6 cm 3 g -1 Below, 1.5 cm 3 g -1 Below, 1.4 cm 3 g -1 Below, 1.3 cm 3 g -1 Below, 1.2 cm 3 g -1 Below, 1.1 cm 3 g -1 less than or equal to 1 cm 3 g-1 The above-described ceria nanoparticles may contain significant amounts of anti-inflammatory, antioxidant, and / or ophthalmic treatment agents within the pore volume range, and may exhibit significant anti-inflammatory, antioxidant, and / or therapeutic effects.

[0078] From another perspective, the present invention relates to a composition for preventing, treating or improving ophthalmic diseases caused by oxidative stress, comprising the mesoporous ceria nanoparticles.

[0079] In an exemplary embodiment, the composition may be used for intravitreal injection.

[0080] In an exemplary embodiment, the composition may be a sustained-release drug release composition.

[0081] In an exemplary embodiment, the ophthalmic disease induced by the oxidative stress may be one or more selected from the group consisting of, but not limited to, cataract, uveitis, retinopathy of prematurity, age-related macular degeneration, primary open-angle glaucoma, corneal aging, keratitis, dry eye, keratoconus, bullous keratopathy, and Fuchs' corneal endothelial dystrophy.

[0082] In one exemplary embodiment, the composition comprises 1 μg mL -1 1000 μg mL -1 , specifically, 1 μg mL -1 Above, 10 μg mL -1 Above, 15 μg mL -1 Above, 20 μg mL -1 Above, 25 μg mL -1 Above, 30 μg mL -1 Above, 35 μg mL -1 Above, 40 μg mL -1 Above, 45 μg mL -1 Above, 50 μg mL -1 Above, 1000 μg mL -1 Below 900 μg mL -1Below 800 μg mL -1 Below, 700 μg mL -1 Below, 600 μg mL -1 Less than or equal to 500 μg mL -1 The composition may include, but is not limited to, mesoporous ceria nanoparticles of the following compositions: 1 μg mL -1 Above, preferably 10 μg mL -1 Ideally, 25 μg mL -1 Above, more preferably 50 μg mL -1 The composition can effectively remove reactive oxygen species by including the mesoporous ceria nanoparticles described above, and the reactive oxygen species removal ability can be increased in a concentration-dependent manner within the above concentration range. In addition, the composition can be used at 1000 μg mL -1 Less than or equal to 500 μg mL, preferably -1 The following mesoporous ceria nanoparticles can be included to minimize side effects such as cytotoxicity that may occur when injected into the eye (eyeball).

[0083] From another aspect, the present invention relates to a method for preventing, treating or improving ophthalmic diseases caused by oxidative stress by applying the mesoporous ceria nanoparticles to a subject.

[0084] In another aspect, the present invention relates to the use of mesoporous ceria nanoparticles for the preparation of a composition for preventing, treating or improving ophthalmic diseases caused by oxidative stress.

[0085] In another aspect of the present invention, the present invention relates to a use of the mesoporous ceria nanoparticles for preventing, treating or improving ophthalmic diseases caused by oxidative stress.

[0086] The present invention relates in another aspect to therapeutic or non-therapeutic uses of mesoporous ceria nanoparticles.

[0087] The present invention relates to a method for producing the mesoporous ceria nanoparticles, comprising the following steps from another aspect.

[0088] (a) a step of mixing a cerium precursor, an imidazole derivative, and 1,1'-Carbonyldiimidazole (CDI) in a non-aqueous solution; and

[0089] (b) A step of obtaining mesoporous ceria nanoparticles from the above mixture.

[0090] In an exemplary embodiment, the cerium precursor may be one or more selected from the group consisting of, but not limited to, cerium(III) acetate hydrate, cerium(III) acetylacetonate hydrate, cerium(III) carbonate hydrate, cerium(IV) hydroxide, cerium(III) fluoride, cerium(IV) fluoride, cerium(III) chloride, cerium(III) chloride heptahydrate, cerium(III) bromide, cerium(III) iodide, cerium(III) nitrate hexahydrate, cerium(III) oxalate hydrate, cerium(III) sulfate, cerium(III) sulfate hydrate, and cerium(IV) sulfate, cerium nitrate, cerium ammonium nitrate, cerium sulfate, cerium phosphate, cerium chloride, cerium carbonate, cerium acetate, and combinations thereof.

[0091] In an exemplary embodiment, the imidazole derivative may be one or more selected from the group consisting of imidazole, 2-methylimidazole, benzimidazole, naphtimidazole, phenanthrimidazole, pyridimidazole, pyrazineimidazole, quinoxalineimidazole, 4-methylimidazole, 4-methyl-2-phenylimidazole, and combinations thereof, but is not limited thereto.

[0092] In an exemplary embodiment, the non-aqueous solution may be one or more selected from the group consisting of, but not limited to, acetone, xylene, toluene, mesitylene, octyl ether, butyl ether, hexyl ether, decyl ether, pyridine, dimethyl sulfoxide, dimethylformamide, octanol, decanol, octane, decane, dodecane, tetradecane, hexadecane, hexane, carbon disulfide, chlorobenzene, dioxolane, tetrahydrofuran (THF), ethylene diamine, dimethyl formamide, dimethyl sulfoxide, ethanol, methanol, and combinations thereof.

[0093] In an exemplary embodiment, step (a) may be performed at, but is not limited to, a temperature of 1°C to 40°C, preferably 10°C to 35°C, and more preferably 15°C to 25°C.

[0094] In an exemplary embodiment, step (a) may be performed for 0.1 to 2.0 hours, preferably 0.5 to 1.5 hours, but is not limited thereto.

[0095] In an exemplary embodiment, step (a) may be stirred using one or more selected from the group consisting of, but not limited to, a magnetic bar, a magnetic stirrer, a rotary mixer, and combinations thereof.

[0096] In an exemplary embodiment, the method may further comprise the step of (c) washing the mesoporous ceria nanoparticles.

[0097] In an exemplary embodiment, the washing step may be performed one or more times using one or more solutions selected from the group consisting of distilled water, acetone, isopropanol, n-propanol, methanol, ethanol, 1-methyl-2-pyrrolidone, and combinations thereof, but is not limited thereto.

[0098] In an exemplary embodiment, the method may further comprise the step of (d) drying the mesoporous ceria nanoparticles.

[0099] In one exemplary embodiment, the method may further comprise the step of (e) loading the ceria nanoparticles with an anti-inflammatory agent, an antioxidant agent, and / or an ophthalmic agent. In addition to the exemplary embodiments described above, additional embodiments may be present in the drawings and detailed description of the invention.

[0100]

[0101] Hereinafter, the composition and effects of the present invention will be described in more detail with examples. However, the examples below are provided for illustrative purposes only to aid understanding of the present invention and are not intended to limit the scope and scope of the present invention.

[0102] [Example]

[0103] [Example 1]

[0104] Preparation of mesoporous cerium oxide nanoparticles (mCeNP)

[0105] 1 mmol Cerium (III) nitrate hexahydrate, 0.5 mmol CDI, and 4 mmol imidazole were added to 25, 12.5, and 12.5 mL of acetone, respectively. The resulting CDI and imidazole solutions were added to a cerium nitrate hexahydrate solution with vigorous stirring at room temperature. After 1 h, the precipitated mCeNPs were collected by centrifugation, washed three times with deionized water, and then dispersed in deionized water.

[0106] [Example 2]

[0107] Characterization of mCeNPs

[0108] Uniform mCeNPs were synthesized at room temperature in acetone solvent using an appropriate molar ratio of reactants (Ce:1,1'-carbonyldiimidazole [CDI]:imidazole = 1:0.5:4) and a reaction time of 1 h without any special equipment, heating, or pressure. The spherical mCeNPs with an average size of approximately 120 nm, composed of small nanoparticles aggregated together, were confirmed using scanning electron microscope (SEM) and transmission electron microscopy (TEM) (Figs. 3 and 4). The hydrodynamic size of mCeNPs was 282.1 ± 9.7 nm, and the zeta potential was confirmed to be 22.4 ± 0.5 mV (Fig. 5). Nitrogen adsorption-desorption isotherm analysis confirmed the presence of mesopores with a size of approximately 2.3 nm in mCeNPs. Additionally, the Brunauer-Emmett-Teller (BET) surface area and Barrett-Joyner-Halenda (BJH) pore volume values ​​of mCeNPs were 102.3 mg, respectively. -1 and 0.08 cm 3 g -1 was confirmed (Fig. 6). The high surface area of ​​the mesopores of mCeNPs can help to tune the catalytic properties, including ROS scavenging ability.

[0109] [Example 3]

[0110] In vitro ROS-scavenging ability of mCeNPs

[0111] The catalase and SOD-mimetic activities of mCeNPs were confirmed using H2O2 / peroxidase and SOD assays, respectively. As shown in Figure 7, the mCeNPs showed a concentration-dependent increase in their reactive oxygen species scavenging ability. In particular, at 100 μg mL -1 mCeNPs showed an H2O2 removal rate of approximately 90% or more, and also showed a high inhibition rate (approximately 90%) in the SOD assay. This indicates that mCeNPs have efficient ROS removal ability due to their mesoporous structure.

[0112] [Example 4]

[0113] In vitro cell viability of mCeNPs

[0114] To investigate the potential cytotoxicity of mCeNPs against retinal tissue and macrophages, in vitro cell viability tests were performed. Various concentrations of mCeNPs were co-cultured with hRPEs and Raw264.7 cells.

[0115] According to the lactate dehydrogenase (LDH) cytotoxicity assay, mCeNPs were <100 μg mL -1 At concentrations of <100 μg mL, mCeNPs did not exhibit significant cytotoxicity against hRPEs, indicating that the mCeNPs used were biocompatible (Fig. 8). In addition, the biocompatibility of mCeNPs against Raw264.7 cells was evaluated using an LDH cytotoxicity assay, which was <100 μg mL -1 No significant cytotoxicity was observed at any concentration (Fig. 8). Even at high concentrations of mCeNPs, cell viability exceeded 80%. This indicates that mCeNPs do not induce significant cytotoxicity in hRPEs and Raw264.7 cells, demonstrating their biocompatibility for bioapplication.

[0116] [Example 5]

[0117] In vitro cytoprotective activity of mCeNPs

[0118] To evaluate the cytoprotective effect of mCeNPs, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was performed under high ROS conditions. As a result, as shown in Fig. 9, after co-culture for 24 h in a medium containing 600 μM H2O2, the viability of hRPEs was approximately 20%. However, in the presence of mCeNPs, cell viability gradually increased as the mCeNPs concentration increased. When co-cultured with mCeNPs for 24 h, cell viability increased from 20% to a maximum of 48%, confirming that cells were protected by the reactive oxygen species-scavenging ability of mCeNPs.

[0119] [Example 6]

[0120] Analysis of the potential of mCeNPs as drug delivery vehicles

[0121] Because mesoporous nanoparticles can serve as potent drug delivery vehicles, the potential of mCeNPs as a drug delivery vehicle was investigated. Indomethacin (IDM), a nonsteroidal anti-inflammatory drug, was loaded onto mCeNPs for 6 h, resulting in a loading efficiency of 12.4 ± 1.5 wt% (Fig. 10). Release profiles revealed an initial burst release of IDM over 12 h, followed by sustained release over 146 h, demonstrating its suitability for sustained-release drug delivery (Fig. 10).

[0122] [Example 7]

[0123] In vitro cell viability of indomethacin-loaded mCeNPs

[0124] To determine the cytotoxicity of indomethacin (IDM)-loaded ceria nanoparticles, an in vitro cell viability test was performed using murine macrophage cells (Raw264.7). As a result, as shown in Fig. 11, the lactate dehydrogenase (LDH) cytotoxicity assay results showed that indomethacin-loaded mCeNPs had a cytotoxicity of <100 μg mL -1 At this concentration, no significant cytotoxicity was observed against the cells, indicating that the indomethacin-loaded mCeNPs have high biocompatibility.

[0125] [Example 8]

[0126] In vitro IL-6 measurement of mCeNPs

[0127] Age-related macular degeneration (AMD) is considered a chronic disease caused by long-term, low-grade inflammation. Increased levels of proinflammatory cytokines, including plasma IL-6, have been reported in AMD patients. IL-6 has been reported to participate in degenerative changes in microglial activity and activation of the signal transducer and activator of transcription 3 (STAT3) signaling pathway. The level of IL-6 may serve as a parameter determining the severity of AMD.

[0128] To further evaluate the anti-inflammatory effects of mCeNPs, Raw264.7 cells were used as an in vitro model. Each mCeNP was administered to the cells and cultured for 6 hours. After 24 hours of LPS stimulation, which induces inflammation and macrophage activation, the level of IL-6 released from Raw264.7 cells into the medium was measured using an enzyme-linked immunosorbent assay (ELISA). As shown in Figure 12, compared with the negative control, LPS stimulation significantly increased IL-6 release. However, Raw264.7 cells pre-incubated with mCeNPs showed a significant decrease in IL-6 expression even under LPS-induced inflammation. Thus, mCeNPs can effectively inhibit IL-6 expression, thereby exhibiting an anti-inflammatory effect.

[0129] In addition, when mesoporous ceria nanoparticles (IDM-mCeNPs) loaded with indomethacin, an NSAID drug, were administered, the amount of IL-6 was confirmed to be reduced by 17.3% compared to particles without drug loading, confirming that loading an anti-inflammatory drug into mCeNPs can produce a synergistic effect in terms of anti-inflammatory effect (Fig. 12).

[0130] [Example 9]

[0131] Confirmation of the in vivo therapeutic effect and biostability of mCeNPs

[0132] The therapeutic potential of mCeNPs with ROS-scavenging activity was confirmed in an AMD mouse model. NaIO3 was used as an oxidant that selectively damages retinal pigment epithelial cells (RPE) through ROS generation. To establish an AMD mouse model, NaIO3 was administered intraperitoneally. As a result, 4 weeks after NaIO3 injection, the outer nuclear layer (ONL) thickness (>50%) was significantly reduced in the NaIO3-treated group, confirming the successful induction of AMD-like pathology (Fig. 13). mCeNPs were administered intravitreally at the early stage of AMD induction. After 4 weeks, the mCeNPs-treated group showed a partial salvage effect with an increase in ONL thickness (14%) (Fig. 13). In other words, mCeNPs partially alleviated the negative effects of NaIO3 on the RPE and preserved the ONL, which contains photoreceptor nuclei. This indicates that mCeNPs exhibit potent ROS scavenging ability and anti-inflammatory effect, contributing to the cytoprotective effect against oxidative stress in RPE.

Claims

1. Mesoporous ceria nanoparticles comprising an anti-inflammatory agent, an antioxidant agent and / or an ophthalmic disease treatment agent.

2. In paragraph 1, The above mesoporous ceria nanoparticles have the ability to remove reactive oxygen species; and anti-inflammatory activity, antioxidant activity, or pharmacological activity.

3. In paragraph 1, The above anti-inflammatory agent is a mesoporous ceria nanoparticle, which is a non-steroidal anti-inflammatory drug (NSAID).

4. In paragraph 1, Mesoporous ceria nanoparticles wherein the antioxidant and / or ophthalmic disease treatment agent is at least one selected from the group consisting of tetramethylpyrazine, resveratrol, ferulic acid, ethyl ferulate (an analogue of ferulic acid) and naringenin, Met 12 and polypeptides consisting of elastin-like polypeptide and αB crystallin, natural flavonoids including luteolin and quercetin, vitamin C, vitamin E, tannic acid, EGCG (Epigallocatechin gallate) and combinations thereof.

5. In paragraph 1, The above ophthalmic disease treatment agent is a mesoporous ceria nanoparticle, which is at least one anti-vascular endothelial growth factor (anti-VEGF) selected from the group consisting of ranibizumab, aflibercept, bevacizumab, brolucizumab, and VABYSMO (faricimab) and combinations thereof.

6. In paragraph 1, Mesoporous ceria nanoparticles having a diameter of 40 nm to 200 nm.

7. In paragraph 1, The surface area of the above ceria nanoparticles is 80 to 200 m 2 g -1 In, mesoporous ceria nanoparticles.

8. In paragraph 1, Mesoporous ceria nanoparticles having a pore size (diameter) of 2 to 50 nm.

9. In paragraph 1, The pore volume of the above ceria nanoparticles is 0.02 to 2.0 cm 3 g -1 In, mesoporous ceria nanoparticles.

10. A composition for preventing, treating or improving ophthalmic diseases caused by oxidative stress, comprising the mesoporous ceria nanoparticles of any one of claims 1 to 9.

11. In paragraph 10, The above composition is a composition for intravitreal injection.

12. In paragraph 10, The above composition is a composition for sustained release drug release.

13. In paragraph 10, A composition wherein the ophthalmic disease caused by the above oxidative stress is at least one selected from the group consisting of cataract, uveitis, retinopathy of prematurity, age-related macular degeneration, primary open-angle glaucoma, corneal aging, keratitis, dry eye, keratoconus, bullous keratopathy, and Fuchs' corneal endothelial dystrophy.

14. In paragraph 10, The above composition is 1-1000μg mL -1 A composition comprising the above mesoporous ceria nanoparticles.

15. A method for producing mesoporous ceria nanoparticles according to any one of claims 1 to 9, comprising the following steps: (a) a step of mixing a cerium precursor, an imidazole derivative, and 1,1'-Carbonyldiimidazole (CDI) in a non-aqueous solution; and (b) A step of obtaining mesoporous ceria nanoparticles from the above mixture.

16. In paragraph 15, A method, wherein the method further comprises the step of (c) washing the mesoporous ceria nanoparticles.

17. In paragraph 15, A method, wherein the method further comprises the step of (d) drying the mesoporous ceria nanoparticles.

18. In paragraph 15, The method further comprises the step of (e) loading an anti-inflammatory agent, an antioxidant agent and / or an ophthalmic disease treatment agent onto the ceria nanoparticles.

Images