NOVEL CERIUM OXIDE NANOCOMPLEX AND A COMPOSITION FOR PREVENTING OR TREATING CEREBRAL INFARCTION 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 brain edema are disclosed. The composition can be used as an efficient nanoparticle therapeutic composition by applying a biocompatible polymer composed of an optimal combination to significantly improve the biomedical stability, biocompatibility, and efficiency of the production process of nanoparticles while maintaining the nanoparticles' excellent inhibitory activity against inflammation. In particular, the composition may be used as an effective therapeutic agent that may help patients with severe cerebral infarction recover their neurological function and greatly improve their survival rate by inhibiting secondary inflammatory response and minimizing tissue injury caused by brain edema.

Claims

1. A method for preventing or treating brain edema, 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, represents a single bond or a double bond, I 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 I 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 brain edema is caused by stroke, cerebral infarction, intracranial hemorrhage, neoplasm of brain, traumatic brain injury, encephalitis, inflammatory brain disease, demyelinating brain disease or intracranial vasculitis.

11. The method of claim 10, wherein the brain edema is caused by cerebral infarction.

12. The method of claim 11, wherein the cerebral infarction is severe cerebral infarction.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0060] FIG. 1 is a diagram illustrating a transmission electron microscope image of a cerium oxide nanocomplex according to the present disclosure.

[0061] 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.

[0062] FIG. 3 is a diagram illustrating a particle size change of the cerium oxide nanocomplex depending on whether or not the compound is present.

[0063] FIG. 4 is a diagram illustrating results of analyzing the particle size change depending on the amounts of polyvinyl pyrrolidone.

[0064] 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.

[0065] 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.

[0066] FIG. 7a and FIG. 7b are diagrams illustrating the evaluation results of hydrogen peroxide and hydroxyl radical scavenging capacity of the cerium oxide nanocomplex.

[0067] FIG. 8 is a diagram illustrating the improvement in survival rate in a mouse model of permanent MCA occlusion by administering the cerium oxide complex of the present disclosure.

[0068] FIG. 9 is a diagram illustrating the results of decreased water content in brain tissue and improved brain edema in a mouse model of permanent MCA occlusion by administering the cerium oxide complex of the present disclosure.

BEST MODE

[0069] 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

[0070] 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.3.6H.sub.2O, 0.540 g, Alfa Aeser, Ward Hill, Mass.) 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

[0071] 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

[0072] 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.3.6H.sub.2O, 0.540 g, Alfa Aeser, Ward Hill, Mass.) 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: The solvent was obtained in the same manner as in the Example 1.

[0073] 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

[0074] 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.

[0075] 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

[0076] 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

[0077] 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

[0078] 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 μmol 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.

[0079] 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).

[0080] 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

[0081] 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 Amplex™ 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 Severe Cerebral Infarction Demonstrated with Improved Survival Rate

[0082] In order to confirm the therapeutic effect of the cerium oxide nanocomplex prepared in the present disclosure for severe cerebral infarction, Sprague-Dawley (SD) rats (Koatech Co., Ltd) were anesthetized with isoflurane to induce a permanent MCA occlusion model. Specifically, the left common carotid artery was tied and fixated with black silk 6-0, external carotid and occipital artery were also tied and fixated with black silk 6-0, and on internal carotid artery hanged black silk 6-0 to create a Y-shaped vessel. Then, the branch point of the Y-shaped vessel was punctured with a 26 G syringe and a filament was inserted through a puncture and advanced around 2 cm into the internal carotid artery to block the middle cerebral artery. Then, with the filament inside, the internal carotid artery was completely tied with black silk, inducing a model of cerebral infarction with permanent MCA occlusion.

[0083] After 1 hour from the time when cerebral infarction was induced, the cerium oxide nanocomplex was intravenously injected into the SD rats at 0.5 mg/kg over 5 minutes, or the same volume of normal saline were injected to the control SD rats. The death or life of the above-mentioned SD rats was checked once a day until 14 days after the time when cerebral infarction was induced (see FIG. 8).

[0084] Referring to FIG. 8, a significant increase in the survival rate was observed in SD rats that were injected with the cerium oxide nanocomplex according to the present disclosure compared to the control group. Specifically, the survival rate of the control group recorded 24.6% (n=31) whereas the group that was administered with cerium oxide nanocomplex showed a survival rate of 49.0% (n=32), which was statistically significant (P=0.03).

Experimental Example 2: Treatment Effect of Cerium Oxide Nanocomplex for Severe Cerebral Infarction Verified Through the Measurement of Water Content in Brain Tissue

[0085] In order to additionally confirm the treatment effect of cerium oxide nanocomplex for severe cerebral infarction prepared in the present disclosure through the measurement of water content in brain tissue, the cerium oxide nanocomplex of the present disclosure was intravenously injected at 0.5 mg/kg over five minutes once after one hour from the time when cerebral infarction was induced in the permanent MCA occlusion model prepared in Experimental Example 1, or the same volume of the normal saline was injected to the control group. At the 72th hour from the time when the cerebral infarction was induced, the brain was harvested and divided into the ipsilateral (left) and contralateral (right) hemispheres. Immediately after the harvest, the wet weight of each cerebral hemisphere was measured and the weight after drying in an oven at 105° C. for 24 hours was measured to calculate the water content in each cerebral hemisphere. The amount of increase in the water content in ipsilateral hemisphere due to brain edema was calculated and evaluated by deducting the water content in the contralateral hemisphere from the one in ipsilateral hemisphere.

[0086] As seen in FIG. 9, the water content increased by 1.29±0.95% (n=9) in SD rats administered with the cerium oxide nanocomplex, and the water content increased by 2.69±0.53% (n=4) (P=0.02) in the control group, confirming that the brain edema in SD rats significantly decreased compared with the control group due to the cerium oxide nanocomplex of the present disclosure.

[0087] 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.