Nanocomplex and method for preparing same

Abstract

Provided are a nanocomplex and a method for preparing same. The nanocomplex comprises a metal ion and a ligand bonded to the metal ion. The method for preparing the nanocomplex comprises the steps of: forming a ligand solution containing a ligand compound; forming a metal precursor solution containing a metal precursor; and mixing the ligand solution and the metal precursor solution.

Description

Nanocomposites and methods for producing them

[0001] The present invention relates to a nanocomposite and a method for producing the same.

[0002] Inorganic nanomaterials composed of metals have significant therapeutic benefits and are being studied as synthetic drugs despite the risk of side effects. Each metal in these inorganic nanomaterials possesses unique electrical properties, resulting in physicochemical functions not found in existing drugs. In particular, metal oxides, which undergo active redox reactions, can act as catalysts in specific biochemical reactions, blocking pathological mechanisms and alleviating the exacerbation of disease symptoms.

[0003] One factor delaying the clinical application of inorganic nanomaterials is their accumulation in the body, driven by the mononuclear phagocytic system (MPS), including the liver and spleen. Dynamic interactions with biomolecules in the body actively alter the surface of injected inorganic nanomaterials, increasing their likelihood of being consumed by phagocytes and other cells. Among the metals captured through this phagocytosis, those that do not undergo metabolic processes in the body are not easily degraded and persist in the body for long periods, posing a risk of chronic toxicity.

[0004] Sepsis is a life-threatening disease worldwide. Each year, 47 to 50 million cases and 11 million sepsis-related deaths are reported, and these numbers continue to rise. Sepsis is characterized by early systemic inflammation caused by bacterial infection, with hyperactivation of innate immune cells and disruption of the vascular endothelial barrier. If left untreated, sepsis can lead to septic shock, multiple organ failure, and even death.

[0005] The present invention provides a nanocomposite having excellent performance.

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

[0007] Nanocomposites according to embodiments of the present invention include a metal ion and a ligand bound to the metal ion.

[0008] The metal ion may include at least one of cerium and iron.

[0009] The ligand may include at least one of DTPA, DOTA, BOPTA, DTPA-BMA, and HP-DO3A. The ligand may be coordinately bonded to the metal ion. The ligand may be multiply bonded to the metal ion. The ligand may be a chelating ligand. The ligand may inhibit oxidation of the metal ion.

[0010] The above nanocomposite can be administered into the human body to treat inflammatory diseases.

[0011]

[0012] Nanocomposites according to embodiments of the present invention include a first nanocomposite and a second nanocomposite. The first nanocomposite includes a first metal ion and a first ligand bound to the first metal ion, and the second nanocomposite includes a second metal ion and a second ligand bound to the second metal ion.

[0013] The first metal ion may include cerium, and the second metal ion may include iron. The first ligand and the second ligand may each include at least one of DTPA, DOTA, BOPTA, DTPA-BMA, and HP-DO3A. The first ligand may be coordinately bonded to the first metal ion, and the second ligand may be coordinately bonded to the second metal ion. The first ligand may be multiply bonded to the first metal ion, and the second ligand may be multiply bonded to the second metal ion. The first ligand and the second ligand may be chelating ligands. The first ligand may inhibit oxidation of the first metal ion, and the second ligand may inhibit oxidation of the second metal ion.

[0014] The above first nanocomposite and the above second nanocomposite can be administered to the human body to treat inflammatory diseases.

[0015]

[0016] A method for manufacturing a nanocomposite according to embodiments of the present invention includes a step of forming a ligand solution containing a ligand compound, a step of forming a metal precursor solution containing a metal precursor, and a step of mixing the ligand solution and the metal precursor solution.

[0017] The mixed solution of the ligand solution and the metal precursor solution can be stabilized at a low pH so that metal nanoparticles are not formed from the metal precursor before chelation by the ligand compound proceeds.

[0018] The ligand chemical compound may include at least one of DTPA, DOTA, BOPTA, DTPA-BMA, and HP-DO3A, and the metal precursor may include at least one of cerium and iron. The ligand compound may be multiply bonded to a metal ion of the metal precursor. The ligand compound may be a chelating ligand compound.

[0019] Nanocomposites according to embodiments of the present invention can exhibit excellent performance. For example, since the nanocomposites are excreted from the body rather than accumulating after administration, they can be utilized for the treatment of inflammatory diseases. In particular, the combined use of nanocomposites containing cerium and nanocomposites containing iron is effective in the treatment of inflammatory diseases (e.g., sepsis).

[0020] Figure 1 shows the structures of CePEG, CeEDTA, and CeDTPA.

[0021] Figure 2 shows the oxygen radical scavenging ability of CePEG, CeEDTA, and CeDTPA.

[0022] Figure 3 shows the XPS analysis results of CePEG, CeEDTA, and CeDTPA.

[0023] Figure 4 shows cell viability after treatment with CePEG, CeEDTA, and CeDTPA.

[0024] Figure 5 shows the cellular uptake rates of CePEG, CeEDTA, and CeDTPA over time.

[0025] Figure 6 shows TEM images of CePEG, CeEDTA, and CeDTPA cultured in aqueous solutions of different compositions.

[0026] Figure 7 shows the extent of cerium accumulation in major organs 24 hours after intravenous injection of CePEG, CeEDTA, and CeDTPA.

[0027] Figure 8 shows the urinary cerium concentration at different times after CeDTPA administration.

[0028] Figure 9 shows the blood cerium concentration at different times after CeDTPA administration.

[0029] Figure 10 shows the active oxygen scavenging ability of FeDTPA.

[0030] Figure 11 shows the degree of hydroxyl radical production of FeDTPA through the Fenton reaction.

[0031] Figure 12 shows the decrease in hydroxyl radical production due to the addition of CeDTPA to FeDTPA.

[0032] Figure 13 shows the oxygen scavenging ability of CF-DTPA of various compositions.

[0033] Figure 14 shows the mechanism applied to the antioxidant synergy effect of CF-DTPA.

[0034] Figure 15 shows the cell viability of RAW 264.7 cells exposed to oxidative stress.

[0035] Figure 16 shows the intensity of hydrogen peroxide detected in RAW 264.7 cells activated with lipopolysaccharide.

[0036] Figure 17 shows a heatmap of differentially expressed genes (DEGs).

[0037] Figure 18 shows a volcano plot of differentially expressed genes.

[0038] Figure 19 shows the results of gene ontology profiling analysis.

[0039] Figure 20 shows a heatmap of representative differentially expressed genes.

[0040] Figure 21 shows the relative changes in gene expression levels of inflammation-related factors (IL-1β and PTGS2) in bone marrow-derived macrophages activated with lipopolysaccharide.

[0041] Figure 22 shows changes in the protein concentrations of IL-1β and TNF-α in bone marrow-derived macrophages activated with lipopolysaccharide.

[0042] Figure 23 shows the survival rate of mice after intravenous injection of various concentrations of CF-DTPA.

[0043] Figure 24 shows the results of a complete blood count analysis of red blood cells (RBCs), white blood cells (WBCs), and platelets (PLTs) after CF-DTPA administration.

[0044] Figure 25 shows the survival rate of septic mice induced by cecal ligation and perforation after administration of CeDTPA, FeDTPA, and CF-DTPA.

[0045] Figure 26 shows the body weight changes in septic mice induced by cecal ligation and perforation after administration of CeDTPA, FeDTPA, and CF-DTPA.

[0046] 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.

[0047] Although terms such as "first" and "second" are used herein to describe various elements, these elements should not be limited by these terms. These terms are used merely to distinguish the elements from one another.

[0048] In the embodiments of the present invention, DTPA (diethylenetriamine pentaacetic acid) is used as a ligand, but is not limited thereto, and DOTA (2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetrazacyclododec-1-yl]acetic acid), BOPTA ((2R)-2-[2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]ethyl-(carboxymethyl)amino]-3-phenylmethoxypropanoic acid), DTPA-BMA (2-[bis[2-[carboxymethyl-[2-(methylamino)-2-oxoethyl]amino]ethyl]amino]acetic acid), HP-DO3A (2-[4,7-bis(carboxymethyl)-10-(2-hydroxypropyl)-1,4,7,10-tetrazacyclododec-1-yl]acetic acid), etc. can be used as a ligand.

[0049]

[0050] Nanocomposites according to embodiments of the present invention include a metal ion and a ligand bound to the metal ion.

[0051] The metal ion may include at least one of cerium and iron.

[0052] The ligand may include at least one of DTPA, DOTA, BOPTA, DTPA-BMA, and HP-DO3A. The ligand may be coordinately bonded to the metal ion. The ligand may be multiply bonded to the metal ion. The ligand may be a chelating ligand. The ligand may inhibit oxidation of the metal ion.

[0053] The above nanocomposite can be administered into the human body to treat inflammatory diseases.

[0054]

[0055] Nanocomposites according to embodiments of the present invention include a first nanocomposite and a second nanocomposite. The first nanocomposite includes a first metal ion and a first ligand bound to the first metal ion, and the second nanocomposite includes a second metal ion and a second ligand bound to the second metal ion.

[0056] The first metal ion may include cerium, and the second metal ion may include iron. The first ligand and the second ligand may each include at least one of DTPA, DOTA, BOPTA, DTPA-BMA, and HP-DO3A. The first ligand may be coordinately bonded to the first metal ion, and the second ligand may be coordinately bonded to the second metal ion. The first ligand may be multiply bonded to the first metal ion, and the second ligand may be multiply bonded to the second metal ion. The first ligand and the second ligand may be chelating ligands. The first ligand may inhibit oxidation of the first metal ion, and the second ligand may inhibit oxidation of the second metal ion.

[0057] The above first nanocomposite and the above second nanocomposite can be administered to the human body to treat inflammatory diseases.

[0058]

[0059] A method for manufacturing a nanocomposite according to embodiments of the present invention includes a step of forming a ligand solution containing a ligand compound, a step of forming a metal precursor solution containing a metal precursor, and a step of mixing the ligand solution and the metal precursor solution.

[0060] The mixed solution of the ligand solution and the metal precursor solution can be stabilized at a low pH so that metal nanoparticles are not formed from the metal precursor before chelation by the ligand compound proceeds.

[0061] The ligand chemical compound may include at least one of DTPA, DOTA, BOPTA, DTPA-BMA, and HP-DO3A, and the metal precursor may include at least one of cerium and iron. The ligand compound may be multiply bonded to a metal ion of the metal precursor. The ligand compound may be a chelating ligand compound.

[0062]

[0063] [Examples and Comparative Examples]

[0064]

[0065] Preparation example of CeDTPA

[0066] 315 mg of DTPA is dissolved in 200 μL of ammonium hydroxide solution and 800 μL of ultrapure distilled water to form a DTPA solution. Cerium (III) chloride is dissolved in ultrapure distilled water to form a 0.4 M cerium chloride solution. 2 mL of the cerium chloride solution is added to the DTPA solution, mixed, and the mixed solution is gently shaken for about 15 minutes to stabilize. The pH of the mixed solution is adjusted to 9 using ammonium hydroxide, and the solution is gently shaken for more than one day, after which the pH of the solution is adjusted to 7 using hydrochloric acid. The CeDTPA thus formed is purified through a desalting column.

[0067]

[0068] Manufacturing example of FeDTPA

[0069] FeDTPA is formed in the same manner as CeDTPA, except that 0.4 M iron(III) chloride solution is used instead of 0.4 M cerium chloride solution.

[0070]

[0071] Preparation example of CF-DTPA

[0072] CeDTPA and FeDTPA are mixed to form CF-DTPA.

[0073]

[0074] Example of manufacturing CeEDTA

[0075] 234 mg of EDTA is dissolved in 200 μL of ammonium hydroxide solution and 800 μL of ultrapure distilled water to form an EDTA solution. Cerium (III) chloride is dissolved in ultrapure distilled water to form a 0.4 M cerium chloride solution. 2 mL of the cerium chloride solution is added to the EDTA solution, mixed, and the mixed solution is gently shaken for about 15 minutes to stabilize. The pH of the mixed solution is adjusted to 9 using ammonium hydroxide, and the solution is gently shaken for more than one day, after which the pH of the solution is adjusted to 7 using hydrochloric acid. The CeEDTA thus formed is purified through a desalting column.

[0076]

[0077] Example of manufacturing CePEG

[0078] 317 mg of cerium(III) acetate hydrate and 4 g of oleylamine are mixed under magnetic stirring. The solution is heated to 90°C until it turns transparent brown. 200 μL of ultrapure distilled water is added to the solution, and the solution is cooled until it turns transparent again. Then, acetone is added to purify the solution. The solution is centrifuged to precipitate ceria nanoparticles, which are then dried. The ceria nanoparticles are dispersed in chloroform to a concentration of 10 mg / mL. 1 mL of PEG solution (10 mg / mL) is added to 0.5 mL of the solution and sonicated to PEGylate the ceria nanoparticles so that PEG encapsulates them. The chloroform is completely evaporated, and the PEGylated ceria nanoparticles (CePEG) are dried and dispersed in ultrapure distilled water.

[0079]

[0080] Ceria (CeO2) nanoparticles are being studied as catalytic antioxidants that alleviate oxidative stress in inflammatory diseases. The active sites of ceria nanoparticles exist on the surface, where oxygen vacancies are generated by weak interatomic bonding energy, and Ce 4+ To Ce 3+ A reduction reaction occurs. The low reduction potential of cerium is Ce 4+ Wow Ce 3+It facilitates electron exchange between ceria and ligands, and it imparts catalytic properties to ceria, enabling it to scavenge reactive oxygen species (ROS). Despite the excellent therapeutic antioxidant and anti-inflammatory properties of ceria nanoparticles, ceria nanoparticles can also accumulate in the mononuclear phagocytic system. Although ceria nanoparticles with a hydrodynamic diameter of less than 5.5 nm have been reported, they are not completely excreted from the body. According to the hard-soft acid-base theory (HSAB theory), cerium is a hard acid and thus forms strong and affinity bonds with hard bases such as oxygen, which are abundant in body fluids in ionic or gaseous form. Therefore, only forms in which the ligand bound to cerium is strongly bound to cerium enough to prevent interactions with biomolecules containing oxygen around cerium can be excreted from the body. Therefore, the correlation between the binding affinity of cerium and ligand and the biological activity of cerium was investigated. To this end, three different cerium-based materials were prepared using three ligands with different binding affinities. Oleylamine was used to synthesize PEGylated ceria nanoparticles (CePEG), while ethylenediaminetetraacetic acid (EDTA) and diethylenetriamine pentaacetic acid (DTPA) were used to synthesize cerium-EDTA (CeEDTA) and cerium-DTPA (CeDTPA) nanocomposites, respectively.

[0081]

[0082] Figure 1 shows the structures of CePEG, CeEDTA, and CeDTPA.

[0083] Referring to Figure 1, CePEG is formed using PEG. Ceria nanoparticles measuring 3 nm can be synthesized using a reverse micelle synthesis method using oleylamine as a ligand. Oleylamine is a monodentate ligand, and the amine group is coordinated to a cerium ion during synthesis. The synthesized ceria nanoparticles are PEGylated using a phospholipid-polyethylene glycol to form CePEG. CePEG has a hydrodynamic diameter of 10 to 20 nm.

[0084] CeEDTA and CeDPTA are prepared using EDTA and DTPA, aminopolycarboxylic acids with strong metal binding affinity. DTPA has a stronger binding affinity for cerium than EDTA due to its additional amine and carboxylate groups. For the synthesis of CeEDTA and CeDTPA, aqueous solutions of EDTA and DTPA are mixed with cerium ions at low pH. It is important to keep the initial pH as low as possible during the synthesis to prevent the formation of ceria nanoparticles before the intended chelation occurs. The ratio of chelator to cerium is maintained at 1:1, and the mixture is gradually increased to pH 9 and stirred for at least one day to prevent precipitation. The mixture is neutralized to pH close to 7 with hydrochloric acid and then purified using a desalting column. The formation of CeEDTA and CeDTPA can be confirmed through mass spectral analysis. The molecular weight of EDTA is approximately 292 g / mol, and the chelating bond of cerium (140 g / mol) replacing a hydrogen atom of EDTA produces a mass spectral peak near 429 g / mol. The sum of the masses of DTPA (393 g / mol) and cerium gives a mass of 532 g / mol. A peak is formed nearby. Another peak is at 553 g / mol. It is formed nearby, indicating that water molecules are additionally formed by combining with the cerium ions of CeDTPA.

[0085]

[0086] Figure 2 shows the oxygen radical scavenging ability of CePEG, CeEDTA, and CeDTPA.

[0087] Referring to Figure 2, CePEG removes superoxide anion, hydrogen peroxide, and hydroxyl radicals to some extent, whereas CeEDTA and CeDTPA do not react with hydrogen peroxide. CeEDTA shows the best performance in removing superoxide anion and hydroxyl radical, and CeDTPA has a lower reactivity toward superoxide anion and hydroxyl radical than CePEG and CeEDTA.

[0088]

[0089] Figure 3 shows the XPS (X-ray Photoelectron Spectroscope) analysis results of Ce 3d of CePEG, CeEDTA, and CeDTPA.

[0090] Referring to Figure 3, cerium in CeEDTA and CeDTPA is Ce 3+ In the case of CePEG, Ce is in most oxidation states. 4+ Peaks corresponding to Ce also appear. Multidentate ligands with strong binding affinity for Ce 3+ Ce of 4+ It appears to limit the oxidation of CeEDTA and CeDTPA. These data allow us to confirm the chemical structures and coordination structures of CeEDTA and CeDTPA in aqueous solutions near neutral pH. Ce in CeEDTA 3+ It forms six coordinate bonds with EDTA and [Ce 3+ (EDTA 4- )] - While forming Ce within CeDTPA 3+ It forms eight coordination bonds with DTPA and [Ce 3+(DTPA 5- )] 2- or [Ce 3+ (DTPA 5- )(H2O)] 2- It forms Ce in aqueous solution 3+ is the inner hydration sphere ([Ce(H2O)9] 3+ ) coordinates with a total of 9 water molecules, DTPA binds most of these nine coordination sites to form a strongly chelated cerium nanocomplex. This strong chelation allows the nanocomplex to maintain structural stability in the in vivo environment. Ce 3+ The relatively strong binding affinity of DTPA hinders the easy electron transfer with superoxide anions and hydroxyl radicals. Thus, the antioxidant effect of cerium-based materials depends not only on the oxidation state of cerium but also on the degree of flexibility of the cerium ion to participate in antioxidant reactions, such as through electron exchange.

[0091]

[0092] Figure 4 shows cell viability after treatment with CePEG, CeEDTA, and CeDTPA, and Figure 5 shows cell uptake rates of CePEG, CeEDTA, and CeDTPA over time.

[0093] Referring to Figures 4 and 5, CeEDTA and CeDTPA exhibit negligible cytotoxicity even at concentrations that reach the acute toxicity of CePEG. The low toxicity of CeEDTA and CeDTPA is attributed to the simple composition of the nanocomposites and the low cellular uptake due to the high hydrophilicity of the chelates. Because CeEDTA and CeDTPA exhibit low toxicity, their reduced antioxidant effect can be compensated for at higher doses, suggesting that CeEDTA and CeDTPA can be used as less toxic superoxide and hydroxyl radical scavengers in systemic inflammatory conditions aggravated by oxidative stress.

[0094]

[0095] Figure 6 shows TEM images of CePEG, CeEDTA, and CeDTPA cultured in aqueous solutions of different compositions. To determine the extent of cerium ion leaching, CePEG, CeEDTA, and CeDTPA were co-cultured in aqueous solution with glutathione (GSH), a biomolecule that typically interacts with xenobiotics, and phosphate buffered saline (PBS).

[0096] Referring to Fig. 6, when cultured in ultrapure distilled water, CePEG is a quasi-spherical ceria nanoparticle with a size ranging from 3 to 5 nm, and no particles are observed in CeEDTA and CeDTPA solutions. When cultured in PBS, CePEG forms a precipitate, and CeEDTA forms insoluble nanorods. There are no visible particles in the CeDTPA solution cultured in PBS. When glutathione (GSH) is added to the solution, the production rate of nanorods formed from CePEG and CeEDTA is further accelerated. When CeDTPA is cultured with glutathione (GSH) and phosphate (PBS), small nanoparticles around 3 nm are observed. Glutathione (GSH) undergoes various chemical reactions with foreign metal substances and contains thiol, amine, and carboxyl functional groups to effectively perform the body's defense mechanism against them. It appears that these characteristics of glutathione (GSH) interfere with the interaction between metals and ligands. DTPA and Ce 3+ The eight coordination bonds between ceria nanoparticles are strong enough to limit reaction with phosphate, but oleylamine, PEG, and EDTA do not bind sufficiently to prevent the formation of insoluble cerium phosphate. This suggests that ceria nanoparticles are biotransformed into cerium phosphate via intracellular mechanisms.

[0097]

[0098] Figure 7 shows the extent of cerium accumulation in major organs 24 hours after intravenous injection of CePEG, CeEDTA, and CeDTPA, Figure 8 shows the concentration of cerium in urine at different times after CeDTPA administration, and Figure 9 shows the concentration of cerium in blood at different times after CeDTPA administration.

[0099] Referring to Figures 7 to 9, most of the injected CePEG remains in the liver and spleen, whereas only a very small amount of injected CeDTPA remains in the body (<1%). Although CeEDTA exists in the form of a small molecular compound that must bypass the mononuclear phagocytosis system and pass through glomerular filtration, leaching of cerium is detected in the liver. These results suggest that even metal nanocomplexes small enough to be excreted in the urine may not be excreted and may accumulate in organs if the interaction between the chelating ligand and the metal ion is not strong enough to withstand exposure to biomolecules. CeDTPA is entirely excreted through the kidneys, and complete excretion takes approximately 4 hours. More than half of the injected CeDTPA is eliminated within 40 minutes after injection, indicating the short half-life of CeDTPA as an antioxidant that can be excreted through the kidneys.

[0100]

[0101] Figure 10 shows the oxygen scavenging ability of FeDTPA, Figure 11 shows the degree of hydroxyl radical production of FeDTPA through the Fenton reaction, Figure 12 shows the decrease in hydroxyl radical production due to the addition of CeDTPA to FeDTPA, and Figure 13 shows the oxygen scavenging ability of CF-DTPA of various compositions.

[0102] Referring to Figures 10 to 13, FeDTPA effectively mimics SOD activity and generates hydroxyl radicals from hydrogen peroxide. When CeDTPA is added to FeDTPA, the amount of hydroxyl radicals decreases as the relative proportion of CeDTPA increases. The optimal ratio of CeDTPA to FeDTPA, which allows for the even removal of representative reactive oxygen species, appears to be 1:3.

[0103]

[0104] Figure 14 shows the mechanism applied to the antioxidant synergy effect of CF-DTPA.

[0105] Referring to Figure 14, both CeDTPA and FeDTPA can scavenge superoxide anions, but only FeDTPA can convert hydrogen peroxide into hydroxyl radicals and water through the Fenton reaction, and CeDTPA can scavenge these hydroxyl radicals, thereby preventing toxic side effects.

[0106]

[0107] Figure 15 shows the cell viability of RAW 264.7 cells exposed to oxidative stress, and Figure 16 shows the intensity of hydrogen peroxide detected in RAW 264.7 cells activated with lipopolysaccharide.

[0108] Referring to Figures 15 and 16, administration of a combination of CeDTPA and FeDTPA (CF-DTPA) can restore cell viability in RAW 264.7 cells exposed to oxidative stress induced by iron sulfate and hydrogen peroxide. The amount of hydrogen peroxide increased in the extracellular matrix of RAW 264.7 cells activated by lipopolysaccharide (LPS) was also significantly reduced after treatment with CF-DTPA.

[0109]

[0110] Figure 17 shows a heatmap of differentially expressed genes (DEGs), and Figure 18 shows a volcano plot of differentially expressed genes. Lipopolysaccharide (LPS)-activated bone marrow-derived macrophages (BMDMs) were cultured with LPS and CF-DTPA for 6 hours, and then next-generation sequencing analysis was performed. The overall expression patterns and Venn diagrams for differentially expressed genes were analyzed between the LPS vs. NT (no-treatment) group and the LPS+CF-DTPA vs. LPS groups.

[0111] Referring to Figures 17 and 18, when analyzing with a change of more than 2-fold set as the threshold and a significance threshold (p-value) of less than 0.05, among the 14,852 genes analyzed, the number of genes that showed differential expression in the LPS vs. NT groups was 4,632, whereas in the LPS+CF-DTPA vs. LPS groups, it was only 531. At the intersection of the Venn diagram, some of the differentially expressed genes were selected and heatmap analysis was performed, resulting in the analysis of a total of 313 genes within the intersection, with 187 genes up-regulated and 126 genes down-regulated. The number of statistically significant up-regulated or down-regulated genes was quantified using a volcano plot in each group.

[0112]

[0113] Figure 19 shows the results of gene ontology profiling analysis. Gene ontology (GO) profiling was performed to identify gene types with significant changes.

[0114] Referring to Figure 19, genes related to protein kinase activity, oxygen saturation, and hypoxia response showed significant differences in the LPS+CF-DTPA group compared to the LPS group.

[0115]

[0116] Figure 20 shows a heatmap of representative differentially expressed genes.

[0117] Referring to Figure 20, the types of representative differentially expressed genes indicate that CF-DTPA affects factors related to inflammation and changes in the concentration of reactive oxygen species.

[0118]

[0119] Figure 21 shows the relative changes in the gene expression levels of inflammation-related factors (IL-1β and PTGS2) in bone marrow-derived macrophages activated with lipopolysaccharide, and Figure 22 shows the changes in the protein concentrations of IL-1β and TNF-α in bone marrow-derived macrophages activated with lipopolysaccharide.

[0120] Referring to Figure 21, changes in the expression levels of interleukin-1β (IL-1β) and prostaglandin-endoperoxide synthase-2 (PTGS-2) are verified through 6-hour and 24-hour qPCR results. While the results are consistent with the next-generation sequencing analysis results at 6 hours, transcript levels decrease in both the LPS and LPS+CF-DTPA groups after 24 hours, unlike after 6 hours.

[0121] Referring to Figure 22, in the LPS group, the protein concentrations of IL-1β and TNF-α did not significantly increase after 6 hours, but a significant increase was observed in both after 24 hours. Another macrophage activation marker, MRC2, was expressed less in the LPS+CF-DTPA group than in the LPS group. These factors were significantly reduced 24 hours after CF-DTPA treatment, supporting the anti-inflammatory effect of CF-DTPA in bone marrow-derived macrophages (BMDMs) activated with lipopolysaccharide (LPS). Treating activated immune cells with CF-DTPA reduced the expression of inflammatory genes in the early treatment period and suppressed subsequent protein expression in the later treatment period, indicating the possibility of a biphasic mechanism of CF-DTPA that can potentially control both the transcriptional and translational stages of the inflammatory response. Furthermore, these analytical results suggest that the short half-life of CF-DTPA may not impair its therapeutic potential, as CF-DTPA can rapidly alter inflammatory gene expression in immune cells, sufficiently influencing protein production after 24 hours.

[0122]

[0123] Although not shown in the figure, experiments were conducted using lipopolysaccharide-activated human umbilical vein endothelial cells (HUVECs) to investigate the effects of CF-DTPA on genetic factors of vascular cells in inflammatory settings. HUVECs were cultured with LPS and CF-DTPA for 6 hours, and then subjected to next-generation sequencing analysis. Of the 16,054 genes, 891 genes showed significant differences in expression between the LPS vs. NT group and 102 genes between the LPS+CF-DTPA vs. LPS groups. Overall heatmaps and Venn diagrams for the LPS vs. NT and LPS+CF-DTPA vs. LPS groups were analyzed. Thirty-six genes at the intersection of the Venn diagrams were additionally visualized using heatmaps. The most prominent change was identified as homophilic cell adhesion mediated by plasma membrane adhesion molecules, a gene ontology term. These proteins, primarily procadherin and cadherin, which form adherens junctions, play a crucial role in vascular permeability. Therefore, these results suggest a potential effect of CF-DTPA on vascular permeability. In sepsis, bloodstream infection leads to vasodilation and damage, which can lead to bacterial translocation and potentially multiple organ damage. This vascular damage can induce excessive infiltration of immune cells, further exacerbating tissue damage. Therefore, reducing vascular permeability may play a key role in mitigating organ damage.To determine whether CF-DTPA could inhibit immune cell recruitment, LPS and CF-DTPA were co-cultured in HUVECs, and the expression levels of representative chemoattractants, C-X-C motif chemokine ligand 1 (CXCL1), C-X-C motif chemokine ligand 10 (CXCL10), and chemokine ligand 2 (CCL2), were evaluated using qPCR at 2, 6, and 24 h after LPS treatment. These factors significantly increased 2 and 6 h after LPS treatment, but were significantly reduced in the CF-DTPA-treated groups. This suggests that CF-DTPA may play a pivotal role in improving the stability of the vascular barrier in inflammatory situations.

[0124]

[0125] Figure 23 shows the survival rate of mice after intravenous injection of various concentrations of CF-DTPA, and Figure 24 shows the results of a complete blood count analysis of red blood cells (RBCs), white blood cells (WBCs), and platelets (PLTs) after CF-DTPA administration.

[0126] Referring to Figures 23 and 24, intravenous injection of 13 mg / kg of CF-DTPA did not affect the survival rate or body weight of mice. In addition, a complete blood count confirmed that CF-DTPA had no acute immunogenicity.

[0127]

[0128] Figure 25 shows the survival rate of septic mice induced by cecal ligation and puncture (CLP) after administration of CeDTPA, FeDTPA, and CF-DTPA, and Figure 26 shows the change in body weight of septic mice induced by cecal ligation and puncture (CLP) after administration of CeDTPA, FeDTPA, and CF-DTPA. Given the short body retention time of CF-DTPA, CF-DTPA was administered via two intravenous injections to optimize the therapeutic effect.

[0129] Referring to Figures 25 and 26, the first administration of CF-DTPA (2 mg / kg) was administered immediately after CLP surgery, and the same dose was administered 3 hours later. In the disease model group, only 17% survived for 12 days, whereas CeDTPA, FeDTPA, and CF-DTPA showed survival rates of 58%, 33%, and 83%, respectively, in septic mice. Co-administration of CeDTPA and FeDTPA significantly increased survival rates, confirming a synergistic effect. The body weights of the surviving mice returned to the normal range, and the CLP group showed the slowest recovery rate.

[0130]

[0131] Twenty-four hours after CLP surgery, plasma from each group was collected to obtain a profile of inflammatory cytokines. In CLP-induced septic mice, proinflammatory cytokines and chemoattractants, such as C-X-C motif ligand 2 (CXCL2), CXCL1, interleukin-6 (IL-6), and chemokine ligand 2 (CCL2), were increased, and administration of CF-DTPA was shown to alleviate this hyperinflammation. Similar trends were also observed in organ tissues such as the liver, spleen, and kidney. Cytokine storm is actually one of the major symptoms of sepsis and is known to induce organ damage and dysfunction. The survival rate of mice increased through the alleviation of inflammation induced by CF-DTPA. Histological analysis of the liver, spleen, and kidney was performed using hematoxylin and eosin (H&E) staining. In the liver, CLP-induced damage, such as congestion and necrosis of hepatic lobules, was found to be alleviated after CF-DTPA administration. The number of areas with high apoptosis in the spleen was quantified by directly counting areas composed primarily of apoptotic cell debris or cells with shrunken nuclei within white and red pulp. CF-DTPA administration reduced the degree of splenic apoptosis and preserved tissue integrity to a certain extent. In the kidneys of mice with CLP-induced sepsis, multiple areas of glomerular shrinkage were observed in the tubules. The CF-DTPA-administered group showed a decrease in the quantified area of ​​apoptosis with glomerular shrinkage compared to the CLP group. Thus, CF-DTPA can function as a non-toxic antioxidant that does not accumulate in the body, reduces blood inflammation levels, protects tissue parenchyma from cell death, and acts as a rapid-acting anti-inflammatory agent.

[0132]

[0133] 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.

[0134] Nanocomposites according to embodiments of the present invention can exhibit excellent performance. For example, since the nanocomposites are excreted from the body rather than accumulating after administration, they can be utilized for the treatment of inflammatory diseases. In particular, the combined use of nanocomposites containing cerium and nanocomposites containing iron is effective in the treatment of inflammatory diseases (e.g., sepsis).

Claims

1. Metal ions; and A nanocomposite comprising a ligand that binds to the above metal ion.

2. In paragraph 1, A nanocomposite characterized in that the metal ion comprises at least one of cerium and iron.

3. In paragraph 1, A nanocomposite characterized in that the ligand comprises at least one of DTPA, DOTA, BOPTA, DTPA-BMA, and HP-DO3A.

4. In paragraph 1, A nanocomposite characterized in that the ligand is coordinately bonded to the metal ion.

5. In paragraph 1, A nanocomposite characterized in that the ligand is multiply bonded to the metal ion.

6. In paragraph 1, A nanocomposite characterized in that the above ligand is a chelating ligand.

7. In paragraph 1, A nanocomposite characterized in that the ligand inhibits oxidation of the metal ion.

8. In paragraph 1, The above nanocomposite is characterized in that it is administered to the human body to treat inflammatory diseases.

9. Containing a first nanocomposite and a second nanocomposite, The first nanocomposite comprises a first metal ion and a first ligand bound to the first metal ion, A nanocomposite characterized in that the second nanocomposite comprises a second metal ion and a second ligand bound to the second metal ion.

10. In paragraph 9, The first metal ion comprises cerium, The second metal ion comprises iron, A nanocomposite, wherein the first ligand and the second ligand each comprise at least one of DTPA, DOTA, BOPTA, DTPA-BMA, and HP-DO3A.

11. In paragraph 9, The first ligand is coordinately bonded to the first metal ion, A nanocomposite characterized in that the second ligand is coordinately bonded to the second metal ion.

12. In paragraph 9, The first ligand is multiply bonded to the first metal ion, A nanocomposite characterized in that the second ligand is multiply bonded to the second metal ion.

13. In paragraph 9, A nanocomposite characterized in that the first ligand and the second ligand are chelating ligands.

14. In paragraph 9, The above first ligand inhibits oxidation of the first metal ion, A nanocomposite characterized in that the second ligand inhibits oxidation of the second metal ion.

15. In paragraph 9, A nanocomposite characterized in that the first nanocomposite and the second nanocomposite are administered to the human body to treat an inflammatory disease.

16. A step of forming a ligand solution containing a ligand compound; A step of forming a metal precursor solution containing a metal precursor; and A method for producing a nanocomposite, comprising the step of mixing the above ligand solution and the above metal precursor solution.

17. In paragraph 16, A method for producing a nanocomposite, characterized in that the mixed solution of the ligand solution and the metal precursor solution is stabilized at a low pH so that metal nanoparticles are not formed from the metal precursor before chelation by the ligand compound occurs.

18. In paragraph 16, The ligand chemical comprises at least one of DTPA, DOTA, BOPTA, DTPA-BMA, and HP-DO3A, A method for producing a nanocomposite, characterized in that the metal precursor comprises at least one of cerium and iron.

19. In paragraph 16, A method for producing a nanocomposite, wherein the ligand compound is multiply bonded to the metal ion of the metal precursor.

20. In paragraph 16, A method for producing a nanocomposite, characterized in that the above ligand compound is a chelate ligand compound.

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