NOVEL CERIUM OXIDE NANOCOMPLEX AND A COMPOSITION FOR PREVENTING OR TREATING LIVER FAILURE COMPRISING THE SAME

Abstract

A cerium oxide nanocomplex, a composition containing the cerium oxide nanocomplex as an active ingredient, and their uses for preventing or treating inflammatory liver disease are disclosed. The present disclosure applies a biocompatible polymer composed of an optimal combination to significantly improve the biomedical stability, biosynthesis and efficiency of the production process of nanoparticles while maintaining the nanoparticles’ excellent inhibitory activity against inflammation. In particular, the present disclosure may be applied as an effective therapeutic composition that effectively controls excessive immune response and inflammatory response and tissue injury in acute liver failure which is a serious condition of rapid loss of liver function that results in multiple organ failure and death.

Claims

1. A method for preventing or treating inflammatory liver disease, comprising administering to a subject in need thereof a cerium oxide nanocomplex comprising: (a) core layer of cerium oxide nanoparticles; and (b) an inner layer including a polymer represented by the following Formula 1: ##STR00003## wherein R.sub.1 and R.sub.2 are each independently hydrogen or oxygen, $\underset{¯}{---}$ represents a single bond or a double bond, l is 1 or 2, and m is an integer of 100 to 1000.

2. The method of claim 1, wherein the cerium oxide nanoparticles are selected from the group consisting of cerium(III) oxide (Ce.sub.2O.sub.3) nanoparticles, cerium(IV) oxide (CeO.sub.2) nanoparticles, and a mixture thereof.

3. The method of claim 1, wherein in Formula 1, R.sub.1 is hydrogen, R.sub.2 is oxygen, and l is 1.

4. The method of claim 1, wherein the nanocomplex further comprises an outer layer that comprises one or more of biocompatible dispersion stabilizers selected from the group consisting of polyglutamic acid (PGA), poly(aspartic acid) (PASP), alginate, poly(acrylic acid) (PAA), poly(methacrylic acid) (PMAA), poly(methyl methacrylic acid), poly(maleic acid) (PMA), poly (butadiene/maleic acid) (PBMA), poly (vinylphosphonic acid) (PSSA), polyvinyl alcohol (PVA) and dextran.

5. The method of claim 4, wherein the biocompatible dispersion stabilizer included in the outer layer is polyglutamic acid (PGA).

6. The method of claim 5, wherein the PGA is poly L-glutamic acid (PLGA).

7. The method of claim 1, further comprising a multifunctional ligand represented by the following Formula 2: ##STR00004## wherein n is an integer of 3 to 7.

8. The method of claim 7, wherein in Formula 2, n is 5.

9. The method of claim 1, wherein the nanocomplex has an average particle size of 5 nm to 100 nm.

10. The method of claim 1, wherein the inflammatory liver disease is selected from the group consisting of viral hepatitis, toxoplasma hepatitis, alcoholic liver disease, toxic liver disease, liver failure, liver abscess, nonspecific reactive hepatitis, liver infarction, hepatic veno-occlusive disease, injury of liver or gallbladder and liver transplantation-related hepatitis.

11. The method of claim 10, wherein the inflammatory liver disease is liver failure.

12. The method of claim 11, wherein the liver failure is acute or subacute liver failure.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0058] FIG. 1 is a diagram illustrating a transmission electron microscope image of a cerium oxide nanocomplex according to the present disclosure.

[0059] FIG. 2 is a diagram illustrating results of analyzing the shape and dispersion of the cerium oxide nanocomplex depending on the properties of the solvent.

[0060] FIG. 3 is a diagram illustrating a particle size change of the cerium oxide nanocomplex depending on whether or not the compound is present.

[0061] FIG. 4 is a diagram illustrating results of analyzing the particle size change depending on the amounts of polyvinyl pyrrolidone.

[0062] FIG. 5 is a diagram illustrating results of analyzing the particle size for each reaction time of the cerium oxide nanocomplex using a dynamic light scattering device.

[0063] FIG. 6a is a diagram showing results of checking whether or not the cerium oxide nanocomplex are coated with a biocompatible dispersion stabilizer using a surface potential analysis device. FIG. 6b illustrates results of analyzing the particle size of the cerium oxide nanocomplex using a dynamic light scattering device.

[0064] FIG. 7a and FIG. 7b are diagrams illustrating the evaluation results of hydrogen peroxide and hydroxyl radical scavenging capacity of the cerium oxide nanocomplex.

[0065] FIG. 8 is a graph showing results of evaluating the toxic stimulation associated with reactive oxygen species after treating hepatocytes (heap-1c1c7) in which the toxic stimulation is induced by pyrogallol with the nanocomplex of the present disclosure for each concentration.

[0066] FIG. 9a, FIG. 9b, and FIG. 9c show the results of measuring ALT (FIG. 9a), AST (FIG. 9b), and liver tissue weight (FIG. 9c) in Sprague-Dawley(SD) rats. The rats were first injected with D-Galactosamine (GaIN) Hydrochlroride and LPS to induce inflammatory irritation. Then nanocomplex of the present disclosure was administered and the corresponding changes in ALT, AST and liver tissue weight were measured. G1, normal rats; G2, positive control group, G3. a group injected with 0.1 mg/kg of cerium oxide nanocomplex, G4, a group injected with 0.5 mg/kg of cerium oxide nanocomplex.

BEST MODE

[0067] Hereinafter, the present disclosure will be described in detail with reference to the following examples. These examples are only for illustrating the present disclosure in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present disclosure.

EXAMPLES

Examples 1: Synthesis of Cerium Oxide Nanocomplex

[0068] A first solution was prepared by dissolving 6-aminohexanoic acid (6-AHA) (0.65585 g, Sigma-Aldrich, St. Louis, MO) in deionized water (30 mL). A third solution was prepared by adding a second solution in which polyvinylpyrrolidone (PVP) (2.0 g, Sigma-Aldrich, St. Louis, MO) was dissolved in ethyl alcohol (25 mL) and heating the second solution to 70° C. in air while stirring the first solution. Meanwhile, a fourth solution was prepared by dissolving cerium(III) nitrate hexahydrate (Ce(NO.sub.3).sub.3.square-solid.6H.sub.2O, 0.540 g, Alfa Aeser, Ward Hill, MA) in ethyl alcohol (50 mL) at room temperature (about 20° C.). Thereafter, a fifth solution was prepared by adding the fourth solution to the third solution. Then, the temperature of the fifth solution was maintained at 70° C. for 2 hours, and then cooled to room temperature (about 20° C.). Through such a process, cerium oxide nanoparticles in which 6-aminohexanoic acid and polyvinyl pyrrolidone were bound to the surface, were obtained (FIG. 1). Next, the cerium oxide nanoparticles were washed three times with acetone to remove unreacted materials.

Example 2: Synthesis Trend of Cerium Oxide Nanocomplex Depending on Solvent

[0069] In order to identify the formation tendency of particles depending on the synthetic solvent, the formation state of the cerium oxide nanocomplex was confirmed by using a 100% aqueous solvent or a 70% ethyl alcohol solvent.

# 100% Aqueous Solvent

[0070] A first solution was prepared by dissolving 6-aminohexanoic acid (0.65585 g, Sigma-Aldrich, St. Louis, MO) in deionized water (30 mL), and a third solution was prepared by adding a second solution in which polyvinylpyrrolidone (2.0 g, Sigma-Aldrich, St. Louis, MO) was dissolved in deionized water (25 mL) during stirring of the first solution, and then heating the second solution to 70° C. in air. Meanwhile, a fourth solution was prepared by dissolving cerium(III) nitrate hexahydrate (Ce(NO.sub.3).sub.3.square-solid.6H.sub.2O, 0.540 g, Alfa Aeser, Ward Hill, MA) in deionized water (50 mL) at room temperature (about 20° C.). Thereafter, a fifth solution was prepared by adding the fourth solution to the third solution. Then, the temperature of the fifth solution was maintained at 70° C. for 2 hours, and then cooled to room temperature (about 20° C.) to obtain cerium oxide nanoparticles in which 6-aminohexanoic acid and polyvinylpyrrolidone were bonded to the surface. Next, the cerium oxide nanoparticles were washed three times with acetone to remove unreacted materials.

# 70% Ethyl Alcohol Solvent

[0071] The solvent was obtained in the same manner as in the Example 1.

[0072] As the result of analyzing the transmission electron microscopy image, it could be confirmed that the cerium oxide nanocomplex synthesized with 100% aqueous solvent was difficult to identify the individual dispersibility of the particles, and was entangled with each other and aggregated like a spider web, whereas the cerium oxide nanocomplex synthesized with 70% ethyl alcohol solvent had high dispersibility of particles (FIG. 2). It was found from the above results that the alcohol solvent is more advantageous than the aqueous solvent in terms of the reaction rate and stable dispersibility of the particles.

Example 3: Structure Formation of Cerium Oxide Nanocomplex Depending on Components of Synthesis Reactant

[0073] In order to confirm a structural change of the nanocomplex depending on whether or not 6-aminohexanoic acid and polyvinyl pyrrolidone is present during a synthesis process, 6-AHA and PVP were sequentially excluded from the synthesis process of Example 1, and finally, the experiment was conducted in a form including all additives. The particle size was analyzed using dynamic light scattering device.

[0074] As a result, it was confirmed that no particles were formed in the absence of 6-AHA, and very large particles of 100 nm or more were formed in the absence of PVP, whereas uniform particles of 10 nm size were synthesized only when both 6-AHA and PVP were added to the synthesis (FIG. 3). Accordingly, it was confirmed that when two compounds, 6-AHA and PVP, were complementarily participated in the synthesis and were applied simultaneously, nanocomplex having a stable size and dispersity as a biomaterial may be efficiently obtained.

Example 4: Change in Formation of Nanocomplex Depending on Amounts of PVP

[0075] In order to confirm the tendency of nanocomplex formation depending on the amounts of PVP serving as a dispersion stabilizer in the present disclosure, the change in the nanocomplex formation was examined by using PVP of various amounts of 1.0, 1.5, 2.0 and 2.5 g in the synthesis process of Example 1. As a result of analyzing the synthesized particle size through dynamic light scattering analysis, it was possible to obtain uniform particles of the smallest size when PVP was used in a dose of 2.0 g (19 mg/ml). Considering the effect of the size of the nanocomplex on dispersity, function and biological stability, it can be said that this is the result of finding for the most suitable synthesis conditions.

Example 5: Size Formation Trend of Cerium Oxide Nanocomplex Depending on Synthesis Time

[0076] In order to identify the particle size and stability of the cerium oxide nanocomplex that may be synthesized through Example 1 for each synthesis retention time, while the reaction of the fifth solution was maintained, samples were taken every 30, 40, 50, 60, 70, 80, 90, and 120 minutes, and the particle size was analyzed using dynamic light scattering device. As a result, it was confirmed that as the reaction time elapsed, the particle size gradually decreased from 30 nm to 5 nm, and each sample collected for each time had a very stable particle size. Therefore, it was confirmed that the method of the present disclosure is a process capable of manufacturing nanoparticles with remarkably excellent uniformity (FIG. 5).

Example 6: Coating of Cerium Oxide Nanocomplex with Biodispersion Stabilizer

[0077] A suspension was prepared by adding 3.5 mg of cerium oxide nanoparticles prepared through the above-mentioned process to 0.8 mL of sodium acetate buffer (2.5 mM). The suspension was mixed with 0.3 .Math.mol of polyglutamic acid (PLGA) (weight average molecular weight: 9,000) or the same weight of polyvinyl alcohol (PVA) (weight average molecular weight: 9,500) and dextran (weight average molecular weight: 6,000) dissolved in 1.2 mL of sodium acetate buffer. The cerium oxide nanocomplex was obtained by stirring the mixture at room temperature for 5 minutes and combining positively charged 6-AHA bound to the surface of the cerium oxide nanoparticles and surface negatively charged the polyglutamic acid, polyvinyl alcohol, dextran, etc., by electrostatic attraction.

[0078] It was confirmed that the cerium oxide nanocomplex had a surface potential value of 5 mV before coating with a biocompatible dispersion stabilizer, and had a charge value of -40 mV after coating with PLGA, and a charge value close to -1 mV after coating with PVA and dextran, respectively. Also, it was confirmed that different biocompatible dispersion stabilizers were coated on the cerium oxide nanoparticles through this method (FIG. 6A).

[0079] It was confirmed that in a dispersing environment of normal saline (an aqueous 0.9% (w/w) sodium chloride solution) that simulates the environment for use in actual biomedical applications, the cerium oxide nanocomplex had a size of 50 to 100 nm before coating with the biocompatible dispersion stabilizer, and the dispersion size was improved from 10 to 50 nm in all conditions using each dispersion stabilizer after coating with the biocompatible dispersion stabilizer (FIG. 6B).

Example 7: Evaluation of Antioxidant Effect of Cerium Oxide Nanocomplex

[0080] In order to determine the antioxidant effect of the cerium oxide nanocomplex coated with different biocompatible dispersion stabilizers, the scavenging of representative reactive oxygen species, hydrogen peroxide (H.sub.2O.sub.2) and hydroxyl radicals (—OH), was investigated. For the analysis of each reactive oxygen species, an AmplexTM Red Hydergen Peroxide/Peroxidase Assay Kit was used for hydrogen peroxide, and hydroxyl radical antioxidant capacity (HORAC) assay was used for hydroxyl radicals. It was confirmed through each analysis that the cerium oxide nanocomplex not only had the function of scavenging various types of reactive oxygen species, but also that this function was proportional to the concentration of the particles (FIG. 7). In addition, it could be confirmed that although the cerium oxide nanocomplex was coated with different kinds of dispersion stabilizers, the ability to scavenge reactive oxygen species was derived from the core particles by successfully scavenging reactive oxygen species.

Experimental Example 1: Treatment Effect of Cerium Oxide Nanocomplex for Infectious Inflammatory Disease - Inflammatory Liver Disease

[0081] In order to investigate the therapeutic effect of cerium oxide nanocomplex for liver cell toxicity caused by inflammation, the degree of hepatotoxicity derived from pyrogallol was measured. In order to evaluate hepatotoxicity, hepatocytes (heap-1c1c7) were treated with pyrogallol to induce toxic stimuli, and the cerium oxide nanocomplex was treated for 1 hour at each concentration to evaluate the toxic stimulus associated with reactive oxygen species. As illustrated in FIG. 8, it was confirmed that the cerium oxide nanocomplex according to the present disclosure significantly reduced pyrogallol-induced hepatotoxicity in a concentration-dependent manner. It was confirmed that the cerium oxide nanocomplex had an excellent therapeutic effect on hepatocellular toxicity caused by inflammation by showing that hepatotoxicity in the group treated particularly with 500 .Math.M of cerium oxide nanocomplex was reduced to a level similar to that of the negative control group.

Experimental Example 2: Treatment Effect of Cerium Oxide Nanocomplex for Acute Liver Failure Verified through Inhibition of Liver Enzymes

[0082] In order to confirm the therapeutic effect of the cerium oxide nanocomplex prepared in the present disclosure for acute liver failure, Sprague-Dawley (SD) rats were used to induce a model of acute liver failure. Specifically, 700 mg/kg of D-Galactosamine hydrochloride (GaIN) and 1 .Math.g/kg of lipopolysaccharide (LPS) were injected into the abdominal cavity of SD rats once. After 130 minutes, 0.1 mg Ce/kg (G3, 12 rats) or 0.5 mg Ce/kg (G4, 12 rats) of the cerium oxide nanocomplex of the present disclosure was intravenously injected with a disposable syringe for around one minute, or the saline of the same volume was intravenously injected into the control group (G2, 12 rats). Also, six rats (G1) were set as a normal group into which any material including GaIN and LPS was not injected. At the 8th hour from the injection of GaIN and LPS, blood from all living animals was collected and put into SST tube which was centrifugated to separate serum. After blood collection was completed, a forensic autopsy was performed on hepatic tissue of all groups. The alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of separate serum were measured using a blood physicochemical analyzer (7180, HITACHI, Japan). Also, the weight of harvested liver tissue was measured and recorded. As a result, it was confirmed that the ALT and AST significantly decreased in rats injected with the cerium oxide nanocomplex compared with those of control group. (FIGS. 9a and 9b). Also, it was confirmed that the weight of livers of rats injected with the cerium oxide nanocomplex was higher than those of control group.

[0083] Specific portions of the present disclosure have been described in detail hereinabove, but it is obvious to those skilled in the art that such a specific description is only an exemplary embodiment, and the scope of the present disclosure is not limited thereto. Therefore, the substantial scope of the present disclosure will be defined by the claims and equivalents thereof.