Iron oxide-ceria nanoparticle, iron oxide-ceria nanostructure and iron oxide-ceria nanocomposite

Abstract

An iron oxide-ceria nanoparticle, an iron oxide-ceria nanostructure and an iron oxide-ceria nanocomposite are provided. The iron oxide-ceria nanoparticle comprises iron oxide and ceria. The iron oxide-ceria nanostructure comprises an inorganic support and a plurality of iron oxide-ceria nanoparticles dispersed in the inorganic support. The iron oxide-ceria nanocomposite comprises: an inorganic support; a plurality of iron oxide-ceria nanoparticles dispersed in the inorganic support; and a polymer layer that encompasses the inorganic support.

Description

Iron oxide-ceria nanoparticles, iron oxide-ceria nanostructures, and iron oxide-ceria nanocomposites

[0001] The present invention relates to iron oxide-ceria nanoparticles, iron oxide-ceria nanostructures, and iron oxide-ceria nanocomposites.

[0002] Inflammatory bowel disease (IBD), a chronic inflammatory disorder that includes Crohn's disease and ulcerative colitis, is rapidly increasing in both developed and developing countries. IBD is characterized by symptoms such as chronic diarrhea, colonic bleeding, and acute colitis, severely reducing patients' quality of life. The pathogenesis of IBD is complex and complex, involving genetic factors, immune responses, gut microbiota imbalances, and environmental factors. It remains a challenging disease to treat. Existing treatments for IBD include corticosteroids, nonsteroidal anti-inflammatory drugs, anti-TNF-α antibodies, immunosuppressants, and surgery. However, these treatments have drawbacks, including side effects due to nonspecific immunosuppression, treatment resistance, recurrent relapses, and an increased risk of colon cancer.

[0003] The present invention provides iron oxide-ceria nanoparticles having excellent performance.

[0004] The present invention provides an iron oxide-ceria nanostructure having excellent performance.

[0005] The present invention provides an iron oxide-ceria nanocomposite having excellent performance.

[0006] Other objects of the present invention will become apparent from the following detailed description and the accompanying drawings.

[0007] Iron oxide-ceria nanoparticles according to embodiments of the present invention include iron oxide and ceria.

[0008] The iron oxide-ceria nanostructure according to embodiments of the present invention comprises an inorganic support and a plurality of iron oxide-ceria nanoparticles dispersed in the inorganic support.

[0009] An iron oxide-ceria nanocomposite according to embodiments of the present invention comprises an inorganic support, a plurality of iron oxide-ceria nanoparticles dispersed in the inorganic support, and a polymer layer surrounding the inorganic support.

[0010] Iron oxide-ceria nanoparticles, iron oxide-ceria nanostructures, and iron oxide-ceria nanocomposites according to embodiments of the present invention exhibit excellent performance. For example, inflammatory bowel disease can be effectively treated using the iron oxide-ceria nanoparticles, iron oxide-ceria nanostructures, and iron oxide-ceria nanocomposites.

[0011] The iron oxide-ceria nanoparticles exhibit enhanced nanoenzyme activity and biocompatibility compared to conventional ceria nanoparticles, and can effectively remove reactive oxygen species (ROS) and alleviate inflammatory responses. Furthermore, the iron oxide-ceria nanoparticles exhibit lower cytotoxicity than conventional ceria nanoparticles and exhibit high biocompatibility in macrophages and intestinal epithelial cells.

[0012] The above iron oxide-ceria nanostructures have reduced nonspecific cell penetration as the size of the nanoparticles increases, thereby reducing accumulation in the body and allowing them to be naturally excreted.

[0013] The above iron oxide-ceria nanocomposite is coated with a pH-responsive enteric polymer, protecting it from the stomach and ensuring safe delivery to the intestines. The enteric coating prevents the iron oxide-ceria nanocomposite from coagulating and rapidly decomposing in the stomach, allowing it to be slowly released from the intestines, thereby enhancing its therapeutic efficacy. The iron oxide-ceria nanocomposite overcomes the limitations of existing ceria nanoparticles, exhibits high biocompatibility and therapeutic efficacy without accumulation in the body, and can be applied not only to the treatment of inflammatory bowel disease but also to various chronic inflammatory diseases such as arthritis, neuroinflammation, and sepsis.

[0014] FIG. 1 illustrates iron oxide-ceria nanoparticles and iron oxide-ceria nanocomposites according to embodiments of the present invention.

[0015] Figure 2 shows the TEM image and EDS analysis results of iron oxide-ceria nanoparticles.

[0016] Figure 3 shows an SEM image of an iron oxide-ceria nanocomposite.

[0017] Figure 4 shows the Ce measured by ICP-AES from the dialysate of iron oxide-ceria nanocomposite and CF@CaP nanocluster in simulated gastric fluid. 3+ Indicates concentration.

[0018] Figure 5 shows the anti-inflammatory enzyme-mimetic ability of iron oxide-ceria nanoparticles.

[0019] Figure 6 shows Live / Dead fluorescence images obtained from transwell co-cultures of IEC-18 and bone marrow-derived macrophages before and after LPS stimulation and CF nanoparticle treatment.

[0020] Figure 7 shows the corrected total cell fluorescence (CTCF) percentage of living cells.

[0021] Figure 8 shows the secretion of TNF-α, IL-1β, IL-6, IL-4, IL-10, and TGF-β in bone marrow-derived macrophage cultures.

[0022] Figure 9 shows the colon length of the control and treatment groups 9 days after treatment with iron oxide-ceria nanocomposite.

[0023] Figure 10 shows the daily body weight changes of the control and treatment groups during the treatment period.

[0024] Figure 11 shows the daily diarrhea scores of the control and treatment groups.

[0025] Figure 12 shows the blood stool scores of the control and treatment groups.

[0026] Figure 13 shows the gastrointestinal transit of iron oxide-ceria nanoparticles, CF@CaP nanoclusters, and iron oxide-ceria nanocomposites after oral administration.

[0027] Figure 14 shows the biodistribution of Ce in the lung, heart, kidney, spleen, liver, intestine, and stomach lysates in the control and treatment groups.

[0028] Fig. 15 is Ca 2+ Confocal fluorescence images of mouse colon tissue stained with minerals are shown. Green is Ca 2+ The color represents the mineral, the red represents the nucleus, and the arrows represent the residual CF@CaP nanoclusters.

[0029] Figure 16 shows images of H&E (hematoxylin & eosin), anti-iNOS, anti-CD68, anti-Ly6, anti-Arg1, and anti-ZO-1 staining of colon tissues collected from the control and treatment groups of DSS-induced colitis.

[0030] Figure 17 shows the quantified expression ratios of ZO-1, iNOS, and COX2 relative to β-actin in colon tissue lysates through immunoblot analysis.

[0031] Figure 18 shows the expression levels of IL-1β, TGF-β, and IL-10 in colon tissue quantified by ELISA (Enzyme-Linked Immunosorbent Assay).

[0032] Hereinafter, the present invention will be described in detail through examples. The objectives, features, and advantages of the present invention will be readily understood through the following examples. The present invention is not limited to the examples described herein and may be embodied in other forms. The examples introduced herein are provided to ensure that the disclosure is thorough and complete and to sufficiently convey the spirit of the present invention to those skilled in the art. Therefore, the present invention should not be limited by the following examples.

[0033] The sizes of elements in the drawings, or the relative sizes between elements, may be exaggerated to facilitate a clearer understanding of the present invention. Furthermore, the shapes of elements depicted in the drawings may vary slightly due to variations in the manufacturing process, etc. Therefore, unless otherwise specified, the embodiments disclosed herein should not be limited to the shapes depicted in the drawings, and should be understood to include certain variations.

[0034]

[0035] Iron oxide-ceria nanoparticles according to embodiments of the present invention include iron oxide and ceria.

[0036] The molar ratio of cerium and iron in the above iron oxide-ceria nanoparticles may be 3:7.

[0037] The above iron oxide-ceria nanoparticles may have a size of 2 to 4 nm.

[0038] The above iron oxide-ceria nanoparticles can be encapsulated with phospholipid-PEG, and the hydrated size of the encapsulated iron oxide-ceria nanoparticles can be 15 nm.

[0039] The above iron oxide-ceria nanoparticles can be prepared by adding oleylamine to a mixture of an iron oxide precursor and a ceria precursor.

[0040]

[0041] The iron oxide-ceria nanostructure according to embodiments of the present invention comprises an inorganic support and a plurality of iron oxide-ceria nanoparticles dispersed in the inorganic support.

[0042] The above inorganic support may include calcium phosphate.

[0043] The above iron oxide-ceria nanostructure may have a size of 100 nm or more.

[0044] The above iron oxide-ceria nanostructure can be manufactured by agglomerating a plurality of iron oxide-ceria nanoparticles with an inorganic compound.

[0045] The above iron oxide-ceria nanostructure is a solution containing the plurality of iron oxide-ceria nanoparticles and Ca 2+ After mixing the storage solution, PO4 3+ It can be manufactured by adding a storage solution.

[0046]

[0047] An iron oxide-ceria nanocomposite according to embodiments of the present invention comprises an inorganic support, a plurality of iron oxide-ceria nanoparticles dispersed in the inorganic support, and a polymer layer surrounding the inorganic support.

[0048] The molar ratio of cerium and iron in the above iron oxide-ceria nanoparticles may be 3:7.

[0049] The above iron oxide-ceria nanoparticles may have a size of 2 to 4 nm.

[0050] The above inorganic support may include calcium phosphate.

[0051] The inorganic support having the plurality of iron oxide-ceria nanoparticles dispersed therein may have a size of 100 nm or more.

[0052] The above inorganic support can be manufactured by agglomerating an inorganic compound with the plurality of iron oxide-ceria nanoparticles.

[0053] The polymer layer may surround a plurality of inorganic supports.

[0054] The polymer layer may comprise a pH-responsive polymer.

[0055] The above iron oxide-ceria nanocomposite may have a tablet form.

[0056] The above iron oxide-ceria nanocomposite can function as an enteric therapeutic agent.

[0057]

[0058] [Synthesis example of iron oxide-ceria nanoparticles]

[0059] Iron oxide-cerium nanoparticles with various iron and cerium compositions can be synthesized via a non-hydrolyzable sol-gel reaction. Iron(III) acetylacetonate and cerium(III) acetylacetonate hydrate are mixed in a specified molar ratio to prepare 1.5 g of the mixture. For example, to synthesize CF30, 0.52 g of cerium(III) acetylacetonate and 0.98 g of iron(III) acetylacetonate hydrate are used. 30 mL of oleylamine is added to the mixture, stirred at 40°C for 30 minutes, and then heated at a rate of 2°C / min to 90°C. The reaction mixture is aged for 8 hours, forming a colloidal dispersion ranging in color from light to dark brown, depending on the relative compositions of iron and cerium. After cooling the reaction mixture to room temperature, it is washed with an excess of acetone and centrifuged (approximately 10,000 rpm, 20 minutes) to obtain a precipitate of iron oxide-ceria nanoparticles. The resulting iron oxide-ceria nanoparticles are redispersed in chloroform to prepare a colloidal dispersion with a concentration of 20 mg / mL and stored.

[0060]

[0061] [Example of synthesis of ceria nanoparticles]

[0062] Add 15 mL of xylene to a mixture of 1 mmol (0.4 g) of cerium (III) acetate hydrate and 12 mmol (3.2 g) of oleylamine. Heat the mixture at 40°C with stirring for 30 minutes, then increase the temperature at a rate of 2°C / min to 90°C. Once 90°C is reached, rapidly inject 1 mL of deionized water into the reaction mixture and vigorously stir. The reaction mixture is maintained at 90°C for 3 hours, and a yellow colloidal dispersion is formed. The resulting dispersion is cooled to room temperature, washed with 100 mL of acetone, and centrifuged (approximately 10,000 rpm for 20 minutes) to obtain a ceria nanoparticle precipitate. Redisperse the ceria nanoparticles in chloroform to prepare a colloidal dispersion with a concentration of 20 mg / mL and store.

[0063]

[0064] [Example of Synthesis of Iron Oxide-Ceria Nanoparticles and Ceria Nanoparticles Encapsulated in Phospholipid-Polyethylene Glycol]

[0065] Phospholipid-polyethylene glycol can disperse iron oxide-ceria nanoparticles and ceria nanoparticles encapsulated with oleylamine in aqueous conditions. 30 mg of mPEG(2000)-PE is dissolved in 3 mL of chloroform and added to 20 mg of iron oxide-ceria nanoparticles or ceria nanoparticles dispersed in chloroform. The mixture is gently sonicated, the chloroform is removed using a rotary evaporator, and the mixture is dried in a vacuum oven heated to 70°C for 2 hours to remove any residual chloroform. 5 mL of deionized water or PBS is added to the dried mixture, and the mixture is sonicated to obtain a clear colloidal dispersion. Excess mPEG(2000)-PE is washed with deionized water or PBS and removed using ultracentrifugation (approximately 15,000 rpm for 15 minutes) using a 0.4 μm filter. The resulting colloidal dispersion is adjusted to a concentration of 10 mg / mL with deionized water or PBS.

[0066]

[0067] [Synthesis example of iron oxide-ceria nanoparticles conjugated with Cy5.5-NHS ester]

[0068] Amine-functionalized phospholipid-PEG is used together with mPEG(2000)-PE to conjugate Cy5.5 fluorescent dye to the surface of iron oxide-ceria nanoparticles. 27 mg of mPEG(2000)-PE and 3 mg of DSPE-PEG(2000)-amine are dissolved together in 3 mL of chloroform and added to 20 mg of iron oxide-ceria nanoparticles or ceria nanoparticles dispersed in chloroform. The mixture is gently sonicated, concentrated using a rotary evaporator, and incubated in a vacuum oven at 70°C for 2 h to completely remove the chloroform. The dried mixture is redispersed in 5 mL of deionized water to form a transparent colloidal dispersion. 5 mg of Cy5.5 is added to this dispersion and shaken at room temperature for 12 h. Excess Cy5.5 and phospholipid-PEG are removed by ultracentrifugation (approximately 15,000 rpm, 15 min) using a 0.4 μm filter and washing, and a sufficient amount of deionized water is added during the washing process. The colloidal dispersion containing nanoparticles conjugated with fluorescent dyes is adjusted to a concentration of 5 mg / mL with deionized water.

[0069]

[0070] [Synthesis example of iron oxide-ceria nanostructures]

[0071] Iron oxide-ceria nanoparticles are aggregated with calcium phosphate (CaP) using flash precipitation method. Ca 2+ Precursor and PO4 3- Prepare two stock solutions of the precursor, each with a final concentration of 35 mM by dissolving CaCl2 and Na2HPO4 in 0.15 M Tris buffer. 2 mL of the iron oxide-ceria nanoparticle solution dispersed in PBS (10 mg / mL) was added to 5 mL of Ca under strong stirring. 2+ After mixing with the storage solution, 5 mL of PO4 3+The stock solution is immediately added. The precipitation reaction is stopped by stirring for 1 minute, then adding an excess of distilled water. The CF@CaP nanoclusters are separated through gentle centrifugation at 1000 rpm. This produces an iron oxide-ceria nanostructure comprising multiple iron oxide-ceria nanoparticles dispersed and aggregated on an inorganic support, calcium phosphate (CaP).

[0072]

[0073] [Synthesis example of iron oxide-ceria nanocomposites]

[0074] CF@CaP nanoclusters (iron oxide-ceria nanostructures) are coated with a polymer using a modified spin-coating method. 100 g of the coating polymer (Eudragit®S100) is mixed with 1.51 g of triethyl citrate and 0.52 g of talc in 100 mL of distilled water and stirred for 2 hours. 0.2 g of this polymer mixture is added to 0.5 g of CF@CaP nanoclusters and reacted with vigorous stirring for 15 minutes. The resulting suspension is gently centrifuged at 1,000 rpm, and the resulting precipitate is recovered and vacuum-dried at 40°C for 8 hours. Thus, an iron oxide-ceria nanocomposite is prepared.

[0075]

[0076] [Iron oxide-ceria nanoparticles and iron oxide-ceria nanocomposites]

[0077] FIG. 1 shows iron oxide-ceria nanoparticles and iron oxide-ceria nanocomposites according to embodiments of the present invention, FIG. 2 shows a TEM image and EDS analysis results of iron oxide-ceria nanoparticles, and FIG. 3 shows a SEM image of iron oxide-ceria nanocomposites.

[0078] Referring to FIGS. 1 to 3, iron oxide-ceria nanoparticles were synthesized through a non-hydrolyzable sol-gel reaction, and monodispersed, uniform nanoparticles with a size of 2 to 4 nm were obtained. Iron oxide-ceria nanoparticles containing various molar ratios of Fe and Ce can be synthesized, and iron oxide-ceria nanoparticles are abbreviated as CF followed by a number indicating the average molar ratio of Ce. For example, CF90 represents iron oxide-ceria nanoparticles having a molar ratio of Ce:Fe = 90:10. Iron oxide-ceria nanoparticles can be encapsulated with phospholipid-PEG to have higher compatibility and durability under physiological conditions, and their hydrated size is about 15 nm due to the small size of the nanoparticle core.

[0079] To enhance the biosafety of orally administered iron oxide-ceria nanoparticles, synthesized CF30 nanoparticles were aggregated with CaP mineral to form CF@CaP nanoclusters (iron oxide-ceria nanostructures). This minimizes cerium accumulation in the body by eliminating size-dependent nonspecific absorption and mimics endogenous CaP nanoprecipitates, thereby maintaining the drug payload stability of iron oxide-ceria nanoparticles in the intestinal lumen and epithelium. Considering the size-dependent transport of enteric nanoparticles, oral ingestion of small nanoparticles (<10 nm) may increase the risk of nonspecific tissue penetration through transcytosis and subsequent tissue accumulation. In contrast, CF@CaP nanoclusters with a size of ≥100 nm can minimize such nonspecific effects. Except for the relatively rare phagocytosis of specialized microparticle (M) cells, which preferentially uptake nanoparticles with a diameter of 100–500 nm, large CF@CaP nanoclusters can avoid most of the passive diffusion pathways across the gastrointestinal tract (GI tract) and into the central lymphatics. Furthermore, the physical barrier of CaP prevents the corrosion of nanoparticles upon continuous exposure to physiological fluids such as saliva, gastric juice, and intestinal fluid, thereby minimizing the leaching of cerium element from the nanoparticle clusters.

[0080] Iron oxide-ceria nanocomposite (CFNT) can have improved biosafety through clustering. CF@CaP nanoclusters have a wide size distribution of approximately 200 nm and can contain multiple iron oxide-ceria nanoparticles using a flash precipitation method. The CF@CaP nanoclusters are coated with a pH-responsive polymer. For example, the pH-responsive polymer can include a poly(methacrylate)-poly(methyl methacrylate) (PMA-PMMA) copolymer. The nanoclusters encapsulated in the polymer layer using a modified spin-coating method can form an enteric formulation of the iron oxide-ceria nanocomposite (CFNT, CF@CaP@Poly). In Fig. 1, the iron oxide-ceria nanocomposite (CFNT) has a tablet form, but is not limited thereto.

[0081] Figure 4 shows the Ce measured by ICP-AES from the dialysate of iron oxide-ceria nanocomposite and CF@CaP nanocluster in simulated gastric fluid. 3+ The concentration is shown. The protective ability of the polymer layer can be confirmed by exposing iron oxide-ceria nanocomposite (CFNT) and CF@CaP nanoclusters to simulated gastric fluid.

[0082] Referring to Fig. 4, CF@CaP nanoclusters were gradually dissolved in an acidic environment, and cerium ions were saturated in the supernatant over time, but iron oxide-ceria nanocomposite (CFNT) coagulated upon contact with acid, and no significant cerium release was detected in the inductively coupled plasma atomic emission spectroscopy (ICP-AES) analysis.

[0083] Further investigation into the protective effect of the pH-responsive enteric coating revealed that the structural morphology, elemental composition, and electrical potential of CF@CaP nanoclusters within iron oxide-ceria nanocomposites (CFNTs) were preserved even after exposure to simulated gastric fluid. Furthermore, the enteric coating played a role in maintaining the nanoenzyme activity of the iron oxide-ceria nanoparticles.

[0084]

[0085] Enzyme-mimicking ability and physicochemical properties of iron oxide-ceria nanoparticles

[0086] Figure 5 shows the anti-inflammatory enzyme-mimetic ability of iron oxide-ceria nanoparticles.

[0087] Referring to Fig. 5, CF nanoparticles exhibit antioxidant properties equivalent to or superior to those of conventional ceria-based nanoparticles in an in vitro enzyme mimicking assay. By introducing iron (Fe) into ceria nanoparticles, the catalytic activity of CF nanoparticles was enhanced compared to that of conventional ceria nanoparticles, and the nanoenzyme performance was optimal at CF30 (Ce:Fe = 30:70) in superoxide dismutase (SOD), catalase (CAT), and hydroxyl radical antioxidant capacity (HORAC) assays. However, when the molar ratio of Ce in the nanoparticles is less than 30%, the catalytic activity decreases rapidly.

[0088] Physicochemical analysis of CF nanoparticles can explain the catalytic behavior of nanoparticles with an optimized relative molar ratio of Ce and Fe. Since the catalytic properties of ceria-based nanoparticles are mainly determined by the electronic state ratio and reversibility of Ce cations, the electronic states of CF nanoparticles were investigated. XPS analysis results showed that a rapid change in the characteristic Ce peak occurred as the Fe content in CF nanoparticles increased. Representative Ce 3+ The characteristic peaks (885 eV and 902.4 eV) remained distinct up to CF10, but Ce 4+ The characteristic peaks (882.7 eV, 899.9 eV, 915.8 eV) disappeared rapidly from CF30. In particular, Ce 3+ Wow Ce 4+ Relative changes in properties are from CF30 to Ce 3+ Ce 3d of 3 / 2 and Ce 3d 5 / 2It shows that Ce is the optimal point where the peak is prominent. 3+ Since the relative abundance of Ce:Fe plays a key role in forming oxygen vacancies, which are important for the regenerative catalytic activity of ceria-based nanoparticles, the relative molar ratio of Ce:Fe = 30:70 in CF30 nanoparticles is believed to provide the optimal electronic environment for Ce ions to transition between the two oxidation states. Looking at the electronic states of Fe ions, it was confirmed that the representative Fe 2p peaks (709.8 eV and 723.7 eV) developed as the Fe content increased. However, the disappearance of the satellite peak at 718.7 eV indicates that the phase of Fe oxide has a structure closer to γ-F2O3 when the Fe content is low and closer to Fe3O4 when the Fe content is high.

[0089] XRD pattern analysis of CF nanoparticles showed that CF nanoparticles with low Fe content maintained the characteristic cubic fluorite structure of CeO2. However, as the Fe content increased, most of the characteristic peaks of fluorite CeO2 or magnetite Fe3O4 disappeared or broadened, suggesting that Fe-rich CF nanoparticles may have low crystallinity as observed in other Ce-Fe double metal oxides. The introduction of Fe into cerium oxide may decrease the crystallinity of nanoparticles, but as the crystallinity decreases, more vacant sites are formed, which leads to the formation of Ce 3+ The ratio can increase. These physicochemical analysis results explain why CF30 has optimal catalytic performance and provide a basis for CF30's suitability for biomedical applications.

[0090]

[0091] Anti-inflammatory Effects of Iron Oxide-Ceria Nanoparticles

[0092] The key link between the ROS-scavenging capacity of ceria-based nanoparticles and their therapeutic efficacy lies in the nanoparticles' ability to shift macrophage behavior from pro-inflammatory to anti-inflammatory. Therefore, the anti-inflammatory effects of CF nanoparticles on macrophages were investigated. Compared with conventional ceria nanoparticles, CF nanoparticles exhibited lower cytotoxicity against bone marrow-derived macrophages and the IEC-18 cell line, indicating that the enhanced biocompatibility of CF nanoparticles, despite their higher nanoenzyme activity, is related to their increased Fe content.

[0093] Figure 6 shows Live / Dead fluorescence images obtained from transwell co-cultures of IEC-18 and bone marrow-derived macrophages before and after LPS stimulation and after CF nanoparticle treatment, Figure 7 shows the percentage of corrected total cell fluorescence (CTCF) of live cells, and Figure 8 shows TNF-α, IL-1β, IL-6, IL-4, IL-10, and TGF-β secretion in bone marrow-derived macrophage cultures. In Figure 6, green represents live cells, and red represents dead cells. To investigate whether the nanoenzyme activity effect of CF nanoparticles leads to the modulation of pro-inflammatory behavior of macrophages, a co-culture model was constructed by dividing bone marrow-derived macrophages and IEC-18 epithelial cells into transwells (pore size: 3.0 μm), and pro-inflammatory stimulation with LPS and interferon-γ (IFN-γ) was applied to this model.

[0094] Referring to Figures 6 to 8, treatment with CF@CaP nanoclusters in an LPS-stimulated environment alleviates the cytotoxicity of pro-inflammatory macrophages. In addition, treatment of bone marrow-derived macrophages stimulated with LPS and interferon-γ with CF@CaP nanoclusters changes the profile of secreted cytokines. The secretion of representative pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, decreases, whereas the secretion of IL-4, IL-10, and TGF-β increases. In particular, in the nanoparticle-treated group, the expression of IL-1β, which induces the IFN-γ-mediated inflammatory pathway, decreases, while at the same time, the expression of IL-10, which promotes IBD regulation and anti-inflammatory responses, increases.

[0095]

[0096] [Therapeutic Efficacy of Iron Oxide-Ceria Nanocomposites in a DSS (Dextran Sodium Sulfate)-Induced Inflammatory Bowel Disease (IBD) Model]

[0097] Figure 9 shows the colon length of the control and treatment groups 9 days after treatment with iron oxide-ceria nanocomposites, Figure 10 shows the daily body weight changes of the control and treatment groups during the treatment period, and Figures 11 and 12 show the daily diarrhea score and bloody stool score of the control and treatment groups, respectively. To determine whether orally administered iron oxide-ceria nanocomposites (CFNTs) can provide a therapeutic effect on IBD, the DSS-induced enteritis model, which severely damages the intestinal barrier, was established as the main in vivo model.

[0098] Referring to Figures 9 to 12, the group treated with iron oxide-ceria nanocomposite (CFNT) significantly maintained colon length. While partial recovery was observed in the groups treated with conventional ceria or CF-based nanoparticles, only the iron oxide-ceria nanocomposite (CFNT) treatment group demonstrated concomitant improvements in body weight recovery and diarrhea and bloody stool scores. CF nanoparticles demonstrated higher therapeutic scores for acute epithelial damage recovery and symptom relief than single-metal ceria nanoparticles.

[0099] Furthermore, the importance of enteric formulations that protect nanoparticle delivery is further emphasized by weight changes and symptom scores. Exposure of pure nanoparticles above can lead to degradation and loss of function, which can lead to minimal therapeutic effects in vivo. Furthermore, considering weight changes, this can even lead to disease exacerbation. CF nanoparticles can alleviate epithelial damage and enteritis symptoms, promoting recovery from inflammatory damage. The enteric protection of iron oxide-ceria nanocomposite (CFNT) ensures that CF nanoparticles maximize bioavailability, catalytic activity, and therapeutic potential.

[0100]

[0101] In vivo pharmacokinetic analysis of iron oxide-ceria nanocomposites

[0102] Figure 13 shows the gastrointestinal transport after oral administration of iron oxide-ceria nanoparticles, CF@CaP nanoclusters, and iron oxide-ceria nanocomposites, Figure 14 shows the biodistribution of Ce in the lung, heart, kidney, spleen, liver, intestine, and stomach lysates in the control and treated groups, and Figure 15 shows the Ca 2+ Confocal fluorescence images of mouse colon tissue stained with minerals are shown. Green is Ca 2+ The color represents the mineral, the red represents the nucleus, and the arrows represent the residual CF@CaP nanoclusters.

[0103] Referring to FIGS. 13 to 15, after administering iron oxide-ceria nanocomposites (CFNT), iron oxide-ceria nanoparticles (CF), and CF@CaP nanoclusters (iron oxide-ceria nanostructures) to each group, the intestinal transport of iron oxide-ceria nanocomposites (CFNT) was investigated more comprehensively in animal experiments and the real-time signal profiles were observed. The fluorescence signals of iron oxide-ceria nanoparticles or CF@CaP nanoclusters appeared in the peritoneal area within 6 hours after administration, whereas the signal of iron oxide-ceria nanocomposites (CFNT) was confined to the stomach. However, after 24 hours after administration, the prominent fluorescence signal in the peritoneal area was confirmed only in mice treated with iron oxide-ceria nanocomposites (CFNT). Subsequently, the ex vivo fluorescence observation of the gastrointestinal tract (GI tract) of each group revealed a more distinct and dramatic difference in the distribution profile due to the enteric coating.

[0104] The fluorescence signals of Cy5.5-labeled iron oxide-ceria nanoparticles and CF@CaP nanoclusters were prominent throughout the gastrointestinal tract at the beginning of administration, whereas the signal of iron oxide-ceria nanocomposite (CFNT) was confined to the stomach. Twelve hours after administration, the signals of iron oxide-ceria nanoparticles and CF@CaP nanoclusters gradually decreased, whereas the signal of iron oxide-ceria nanocomposite (CFNT) began to diffuse along the intestinal tract at a later time point. In all treatment groups, the signal was barely detectable 48 hours after administration. This indicates that the pH-responsive coagulation of iron oxide-ceria nanocomposite (CFNT) in the stomach delayed its peristalsis into the intestine. Indeed, coagulated thin polymer clusters were observed in the lumen of gastric tissue harvested 6 hours after administration. However, upon reaching the intestine, the enteric coating dissolved, allowing the CF@CaP nanoclusters to diffuse continuously. In other words, enteric coating provides additional control over pharmacodynamics. Furthermore, no significant fluorescence signal was detected in any treatment group 48 hours after administration, indicating that most of the iron oxide-ceria nanoparticles present in the gastrointestinal tract were excreted within 2 days after administration.

[0105] Although the intravital observation results alleviated most concerns about the digestion or bioaccumulation of excessive cerium (Ce), the possibility of cerium accumulation following oral administration of iron oxide-ceria nanocomposites (CFNTs) was further investigated. After conducting the experiment in a DSS-induced enteritis model, major organs were harvested from the control and treated groups after 9 days, and residual cerium was measured in the organ lysates using inductively coupled plasma mass spectrometry (ICP-MS). Trace amounts of cerium were detected in the stomach and intestinal lysates, but systemic cerium accumulation in distant organs following oral administration of iron oxide-ceria nanocomposites (CFNTs) was not observed. For more precise analysis, residual CF@CaP nanoclusters in the intestine were localized using calcein-AM staining, which labels mineralized calcium.

[0106] In the lateral cross-section of mouse intestinal tissue from the iron oxide-ceria nanocomposite (CFNT)-treated group, CF@CaP nanoclusters were observed distributed along the intestinal lumen surface. A distinct green fluorescence signal was also observed at the basal region of the intestinal tissue, which may be due to damage to the intestinal barrier at the site of inflammation. Additionally, some CF@CaP nanoclusters, approximately 100 nm in size, may have been spontaneously taken up by gut-specific M cells or tissue-resident phagocytes.

[0107] While the nanoclusters were effectively translocated within the gastrointestinal tract, no acute toxicity or morphological changes in the liver tissue were observed in the CFNT-treated group. Given that cerium (Ce) was not detected in the liver lysate, this suggests that the size-dependent effect of CF@CaP nanoclusters contributed to inhibiting passive diffusion or cell migration from the intestine to the liver or central lymphatic vessels. Because the translocation of CFNT was confined to the intestinal lumen and epithelium, no major signs of systemic toxicity were observed.

[0108]

[0109] In vivo anti-inflammatory modulation of iron oxide-ceria nanocomposites

[0110] Figure 16 shows images of H&E (hematoxylin & eosin), anti-iNOS, anti-CD68, anti-Ly6, anti-Arg1, and anti-ZO-1 staining of colon tissues collected from the control and treatment groups of DSS-induced colitis, Figure 17 shows the quantified expression ratios of ZO-1, iNOS, and COX2 relative to β-actin in colon tissue lysates through immunoblot analysis, and Figure 18 shows the expression levels of IL-1β, TGF-β, and IL-10 in colon tissues quantified by ELISA (Enzyme-Linked Immunosorbent Assay).

[0111] Referring to Figures 16 to 18, histological and immunohistochemical analysis results showed that administration of iron oxide-ceria nanocomposite (CFNT) not only maintained the structural integrity of the colon but also induced an anti-inflammatory effect on DSS-induced enteritis.

[0112] Hematoxylin-eosin (H&E) staining of colon samples from the control and iron oxide-ceria nanocomposite (CFNT)-treated groups revealed that the epithelial layer and luminal structure of the colon were maintained intact when iron oxide-ceria nanocomposite (CFNT) was administered. Immunohistochemical staining results also showed that the iron oxide-ceria nanocomposite (CFNT)-treated group showed a significant decrease in iNOS expression, a decrease in the recruitment of monocytes (CD68) and neutrophils (Ly6), and an increase in the expression of Arg1. This indicates that iron oxide-ceria nanocomposite (CFNT) provided an anti-inflammatory effect on the damaged intestinal barrier in the enteritis model. In addition, Western blot analysis of the colon lysate from the iron oxide-ceria nanocomposite (CFNT)-treated group showed that the expression of iNOS and COX2 decreased and the level of ZO-1 increased, indicating that the iron oxide-ceria nanocomposite (CFNT) contributed to the inhibition of inflammation and the recovery of tight junctions of intestinal epithelial cells.

[0113] In addition, when the cytokine levels in the colon lysate were measured by ELISA, IL-1β decreased, while TGF-β and IL-10 increased simultaneously in the iron oxide-ceria nanocomposite (CFNT) treatment group. The fact that iron oxide-ceria nanocomposite (CFNT) antagonistically regulated the levels of IL-1β and IL-10 serves as a factor that enhances its role in immune regulation in the IBD model, and the simultaneous induction of IL-10 and inhibition of IL-1β production are considered important indicators in the disease alleviation process. In addition, the increased secretion of TGF-β, a key regulator in the immunosuppression and fibrosis recovery process of IBD mediated by Treg cells, also explains why the recovery in the iron oxide-ceria nanocomposite (CFNT) treatment group was improved.

[0114] Although not shown in the figure, IκBα, NF-κB, and pSTAT3 were reduced in the colon lysates of the iron oxide-ceria nanocomposite (CFNT)-treated group, which serves as an initial indication that CF nanoparticles are involved in simultaneously suppressing inflammatory pathways. In addition, H&E staining of spleen tissues showed a decrease in activity in lymphoid tissues, supporting the possibility that iron oxide-ceria nanocomposite (CFNT) treatment may exert systemic anti-inflammatory effects in the enteritis model.

[0115] Proteomic analysis revealed that the therapeutic efficacy of iron oxide-ceria nanocomposites (CFNTs) in DSS-induced enteritis was associated with modulation of the innate immune response. CFNT treatment suppressed or reversed the expression of several cytokines associated with DSS-induced inflammation. This includes decreased expression of monocyte and neutrophil proliferation and differentiation (G-CSF, TREM-1), complement precursors and chemoattractants (C5-C5a, CXCL1 / KC, CCL2 / JE, CXCL12 / SDF-1), pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6), and macrophage stimulating pathway (MIP-1α, MIP-1β). The cytokine profiles observed in the ceria nanocomposite (CeNT)-treated and iron oxide-ceria nanocomposite (CFNT)-treated groups showed contrasting patterns, and the pro-inflammatory state observed in the ceria nanocomposite (CeNT)-treated group indicates that the persistent inflammatory response after DSS-induced epithelial damage is the main cause of weight loss and aggravation of intestinal damage. Therefore, the therapeutic efficacy of iron oxide-ceria nanocomposite (CFNT) is thought to result from the modulation of innate immune activity.

[0116]

[0117] Iron oxide-ceria nanoparticles according to embodiments of the present invention exhibit improved biocompatibility and nanoenzyme activity compared to conventional ceria nanoparticles. The enteric coating of the iron oxide-ceria nanocomposite maintains the efficacy of the nanoparticles and provides pharmacodynamic control, and clustering with CF@CaP enables excretion of the nanoparticles. Orally administered iron oxide-ceria nanoparticle clusters are mostly excreted from the body, with minimal off-target effects. The iron oxide-ceria nanocomposite exhibits therapeutic efficacy by restoring intestinal structure and function and modulating the gut-associated immune system into an anti-inflammatory environment.

[0118]

[0119] We have discussed specific embodiments of the present invention. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than restrictive. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

[0120] Iron oxide-ceria nanoparticles, iron oxide-ceria nanostructures, and iron oxide-ceria nanocomposites according to embodiments of the present invention exhibit excellent performance. For example, inflammatory bowel disease can be effectively treated using the iron oxide-ceria nanoparticles, iron oxide-ceria nanostructures, and iron oxide-ceria nanocomposites.

[0121] The iron oxide-ceria nanoparticles exhibit enhanced nanoenzyme activity and biocompatibility compared to conventional ceria nanoparticles, and can effectively remove reactive oxygen species (ROS) and alleviate inflammatory responses. Furthermore, the iron oxide-ceria nanoparticles exhibit lower cytotoxicity than conventional ceria nanoparticles and exhibit high biocompatibility in macrophages and intestinal epithelial cells.

[0122] The above iron oxide-ceria nanostructures have reduced nonspecific cell penetration as the size of the nanoparticles increases, thereby reducing accumulation in the body and allowing them to be naturally excreted.

[0123] The above iron oxide-ceria nanocomposite is coated with a pH-responsive enteric polymer, protecting it from the stomach and ensuring safe delivery to the intestines. The enteric coating prevents the iron oxide-ceria nanocomposite from coagulating and rapidly decomposing in the stomach, allowing it to be slowly released from the intestines, thereby enhancing its therapeutic efficacy. The iron oxide-ceria nanocomposite overcomes the limitations of existing ceria nanoparticles, exhibits high biocompatibility and therapeutic efficacy without accumulation in the body, and can be applied not only to the treatment of inflammatory bowel disease but also to various chronic inflammatory diseases such as arthritis, neuroinflammation, and sepsis.

Claims

1. Iron oxide-ceria nanoparticles containing iron oxide and ceria.

2. In paragraph 1, In the above iron oxide-ceria nanoparticles, the molar ratio of cerium and iron is 3:7, The above iron oxide-ceria nanoparticles have a size of 2 to 4 nm and are encapsulated with phospholipid-PEG, Iron oxide-ceria nanoparticles, characterized in that the hydration size of the encapsulated iron oxide-ceria nanoparticles is 15 nm.

3. In paragraph 1, The above iron oxide-ceria nanoparticles are, Iron oxide-ceria nanoparticles characterized in that they are prepared by adding oleylamine to a mixture of iron oxide precursor and ceria precursor.

4. Weapon support; and An iron oxide-ceria nanostructure comprising a plurality of iron oxide-ceria nanoparticles dispersed on the above-described inorganic support.

5. In paragraph 4, An iron oxide-ceria nanostructure characterized in that the inorganic support comprises calcium phosphate.

6. In paragraph 4, An iron oxide-ceria nanostructure characterized in that the iron oxide-ceria nanostructure has a size of 100 nm or more.

7. In paragraph 4, The above iron oxide-ceria nanostructure is an iron oxide-ceria nanostructure characterized in that a plurality of iron oxide-ceria nanoparticles are produced by agglomeration with an inorganic compound.

8. In paragraph 7, The above iron oxide-ceria nanostructure is a solution containing the plurality of iron oxide-ceria nanoparticles and Ca 2+ After mixing the storage solution, PO4 3+ An iron oxide-ceria nanostructure characterized in that it is manufactured by adding a storage solution.

9. Weapon support; A plurality of iron oxide-ceria nanoparticles dispersed on the above inorganic support; and An iron oxide-ceria nanocomposite comprising a polymer layer surrounding the above-mentioned inorganic support.

10. In paragraph 9, An iron oxide-ceria nanocomposite characterized in that the molar ratio of cerium and iron in the above iron oxide-ceria nanoparticles is 3:

7.

11. In paragraph 9, An iron oxide-ceria nanocomposite, characterized in that the above iron oxide-ceria nanoparticles have a size of 2 to 4 nm.

12. In paragraph 9, An iron oxide-ceria nanocomposite characterized in that the inorganic support comprises calcium phosphate.

13. In paragraph 9, An iron oxide-ceria nanocomposite, characterized in that the inorganic support on which the plurality of iron oxide-ceria nanoparticles are dispersed has a size of 100 nm or more.

14. In paragraph 9, The above inorganic support is an iron oxide-ceria nanocomposite characterized in that the inorganic compound is produced by agglomeration with the plurality of iron oxide-ceria nanoparticles.

15. In paragraph 9, An iron oxide-ceria nanocomposite characterized in that the polymer layer surrounds a plurality of inorganic supports.

16. In paragraph 9, An iron oxide-ceria nanocomposite, wherein the polymer layer comprises a pH-responsive polymer.

17. In paragraph 9, An iron oxide-ceria nanocomposite characterized in that the iron oxide-ceria nanocomposite has a tablet form.

18. In paragraph 9, An iron oxide-ceria nanocomposite characterized in that the above iron oxide-ceria nanocomposite functions as an enteric therapeutic agent.

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