KR101696903B1 - Pharmaceutical composition for treating intracerebral hemorrhage comprising ceria nanocomposite - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating nontraumatic intracranial hemorrhage comprising ceria nanocomposite. The pharmaceutical composition comprising ceria nanocomposite effectively arrives in cytoplasm through a cell membrane, and shows excellent antioxidant and anti-inflammatory effects on hematoma and hematoma periphery areas, thereby being used for treating nontraumatic intracranial hemorrhage.

Description

[0001] PHARMACEUTICAL COMPOSITION FOR TREATING INTRACEREBRAL HEMORRHAGE COMPRISING CERIA NANOCOMPOSITE [0002]

The present invention relates to a pharmaceutical composition for treating non-traumatic intracranial hemorrhage comprising ceria nanocomposite.

Non-traumatic intracerebral hemorrhage (ICH) is a subtype of stroke, with a mortality rate of about 30%. However, there are few known methods of treating ICH other than current blood pressure reduction.

In theory, it may be considered to remove hematoma, which is the primary cause of brain damage caused by hemorrhage for ICH therapy. Removal of hematoma can be done mostly through surgical operation. However, it is not easy to remove hematoma in clinical practice because hemorrhage may occur deeply in the brain when ICH is developed, and surgical operation is difficult and complication due to surgery may be accompanied.

In addition to the above hematomas, secondary damage of the brain derived therefrom is also a major cause of damage to brain tissue.

Specifically, as the coagulation components such as early hemorrhagic hemoglobin and thrombin are released, secondary damage such as oxidative damage due to reactive oxygen species (ROS), inflammation, edema, and brain tissue necrosis occurs. In cases of low blood loss, most patients show only initial seizures, but subsequent secondary damage can lead to fatal consequences such as dying or missing cranial nerves. In particular, reactive oxygen species including superoxide anion (O ₂ ^- ), hydrogen peroxide (H ₂ O ₂ ), and hydroxyl radical (HO ㆍ) are major causes of brain tissue necrosis, It is a major cause of tissue damage in the periphery.

For this reason, many researchers have made various attempts to reduce secondary brain damage, and recently ceria nanoparticles have been studied as a new therapeutic agent.

In one example, ceria (CeO ₂₎ nanoparticles can reversibly combine with oxygen, and CeO ₂ (Celardo, I., et al., Nanoscale (3), 1411-1420, (2011)) have been reported to have a free radical scavenging activity due to the oxidation state change between Ce ^{3 +} and Ce ^{4 + on} the surface of nanoparticles.

In addition, ceria nanoparticles have been found to have a superoxide dismutase-like activity (when cerium is oxidized to Ce ^< ^{4 + &gt};) and catalase-like activity (cerium reduced to Ce ^< ^{3 +} > to protect cells from excess oxide anions and hydrogen peroxide (Heckert, EG et al., Biomaterials (29), 2705-2709, (2008); and Korsvik, C. et al., Chem . Commun ., 1056-1058, 2007 )).

International Patent Application No. PCT / US2006 / 024963 discloses cerium nanoparticles for therapeutic compositions, which only mention that the composition can be used for the treatment of stroke without specific experimentation, and have been used to treat cerebral hemorrhage, specifically non-traumatic intracranial hemorrhage It is not disclosed at all. Estevez et al. Also disclose that ceria nanoparticles can be used in the treatment of stroke without initiation of treatment for cerebral hemorrhage. (AY Estevez, et al. , Volume: 51, Issue: 6, Pages: 1155 -1163, (2011)).

However, the above prior art discloses only ceria nanoparticles used in stroke, particularly cerebral infarction therapy, and discloses nothing about a complex that includes cerebral hemorrhage, particularly ceria nanoparticles or ceria nanoparticles used in the treatment of ICH It is not.

Cerebral infarction and cerebral hemorrhage are only common in that they are diseases that occur in the brain, and they are different diseases that have completely different reasons and treatment methods. Specifically, cerebral infarction refers to a thrombus formed in blood vessels (small blood vessels, large blood vessels, etc.), resulting in clogged blood flow, resulting in necrosis of the brain due to lack of blood flow. Hypoxic damage occurs in brain tissue that develops in cerebral infarction. These cerebral infarcts are blocked with blood vessels, so they maintain maximum blood pressure to maintain blood flow. The excess fluid is also supplied, and thrombolysis, thrombolysis, or thrombolysis is performed to remove or avoid thrombosis using thrombolytic agents, antithrombotic agents, and anticoagulants.

On the other hand, cerebral hemorrhage is a disease in which the cerebral vessels are ruptured and the brain is contaminated by the blood that flows into the brain tissue. In other words, when cerebral hemorrhage occurs, physical brain tissue damage is caused by hematoma generated primarily by hemorrhage, and inflammation is induced from a cytotoxic substance such as reactive oxygen species, heme, thrombin, A reaction occurs, which causes edema around the hematoma. In this hemorrhagic hemorrhage, it is important to suppress swelling and inflammation around the hematoma. In order to suppress the inflammatory reaction, different treatments must be applied to the cerebral infarction. For example, because hemorrhage is a blood vessel burst, blood pressure lowering is performed to lower blood pressure as much as possible in order to reduce blood flow, or coagulation promoting technique for promoting blood coagulation is attempted.

Accordingly, the present inventors have completed the present invention by developing a pharmaceutical composition for treating intracerebral hemorrhage including ceria nanocomposite having excellent therapeutic effect on hematoma, oxidative damage, inflammation and the like caused by the onset of ICH Respectively.

PCT / US2006 / 024963

 Celardo, I., et al., Nanoscale (3), 1411-1420, (2011)  Heckert, E. G. et al., Biomaterials (29), 2705-2709, (2008)  Korsvik, C. et al., Chem. Commun., 1056-1058, (2007)  A Y Estevez, et al., Free Radical Biology and Medicine, Volume: 51, Issue: 6, Pages: 1155-1163, (2011)

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for treating non traumatic intracranial hemorrhage comprising ceria nanocomposite.

In order to achieve the above object, the present invention provides a pharmaceutical composition for treating non traumatic intracranial hemorrhage comprising ceria nanoparticles, a surfactant, and a ceria nanocomposite comprising a phospholipid-polyethylene glycol (PEG).

The pharmaceutical composition comprising the ceria nanocomposite of the present invention efficiently reaches the cytoplasm through the cell membrane and exhibits excellent antioxidant and antiinflammatory effects in the hematoma and the hematoma surrounding region. Therefore, it is useful for treating non traumatic intracranial hemorrhage Can be used.

FIG. 1 is a graph showing a lactate dehydrogenase assay performed on various concentrations of ceria nanocomposite.
Fig. 2 is a graph showing the removal rates of reactive oxygen species in various concentrations of ceria nanocomposite.
FIG. 3 shows the result of performing hydrogen peroxide analysis (H ₂ O ₂ assay) on various concentrations of ceria nanocomposite.
FIG. 4 shows results of hydrogen peroxide analysis (H ₂ O ₂ assay) after ceria nanocomposite is injected into injured cells and non-damaged cells.
5 is a photograph taken after inducing a grease reaction to measure NO ₂ ^- after ceria nanocomposite is injected.
6 is a graph showing the amount of NO ₂ ^- released (absorbance) with time.
7 is a graph showing quantitative analysis of COX-2 protein expression according to ceria nanocomposite concentration.
FIG. 8 is a Western blot analysis of the COX-2 protein of FIG. 7. FIG.
Fig. 9 is a photograph of a dead cell observed by fluorescence microscope after ceria nanocomposite is injected.
10 is a graph showing quantitative analysis of the dead cells of FIG.
11 is a schematic diagram showing a process of inducing ICH in a rat (Rat).
12 is a graph quantifying the volume of the hematoma.
Fig. 13 is a graph of the brain volume measurement.
14 is a photograph taken by immunofluorescence microscopy after administering ceria nanocomposite to a brain in which intracranial hemorrhage has occurred.
15 is a graph showing quantitative analysis of CD68-positive cells after administration of ceria nanocomposite to a brain in which intracranial hemorrhage occurred.
16 is a graph comparing COX-2 protein expression rates in the case where the ceria nanocomposite is injected and the case where the ceria nanocomposite is not injected.
FIG. 17 is a Western blot analysis of the COX-2 protein of FIG. 16; FIG.
FIG. 18 is a photograph of a brain in which intracranial hemorrhage occurs by fluorescence microscopy after intravenous injection of rhodamine-isothiocyanate (RITC) labeled ceria nanocomposite.
19 is an image reconstructed in two dimensions by approximating the distribution and fluorescence intensities of rhodamine-isothiocyanate (RITC) on brain tissue slides with kernel density.
FIG. 20 is a view showing a combination of the photographs of FIGS. 18 and 19 and reconstructing a three-dimensional image.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for treating non traumatic intracranial hemorrhage comprising ceria nanoparticles, a surfactant, and a ceria nanocomposite composed of a phospholipid-polyethylene glycol (PEG).

The ceria nanoparticles may be surrounded by a surfactant and capped with a phospholipid-PEG.

Specifically, the ceria nanoparticles may be surrounded by a surfactant. Wherein said surfactant is selected from the group _consisting of C _6-20 alkylamines, such as oleylamine, octylamine, hexadecylamine or octadecylamine; Alkyltrimethylammonium salts such as cetyltrimethylammonium bromide, octyltrimethylammonium bromide or dodecyltrimethylammonium bromide; (Ethylene oxide) ₂₀ (propylene oxide) ₇₀ (ethylene oxide) ₂₀ poly (alkylene oxide) tri-co-polymer; And alkali salts of oleic acid, linoleic acid, lauric acid, myristic acid, palmitic acid or stearic acid, C _12-18 fatty acid, and the like. Preferably oleylamine.

The size of the ceria nanoparticles may be from 3 to 6 nm, preferably from 3 to 4 nm. When the ceria nanoparticles are in the above range, they have good dispersibility, small particle size, uniformity and excellent effect.

Furthermore, the ceria nanoparticles surrounded by the surfactant may be in the form of a phospholipid-PEG capped form. The ceria nanocomposite is enclosed by an amphipathic molecule, phospholipid-PEG, which allows biomedical administration. The phospholipid-PEG may have a weight average molecular weight of about 100 to 10,000 Da, depending on the number of ethylene oxides in the polymer chain.

The size of the ceria nanocomposite may be 100 nm or less and specifically 5 to 100 nm, 5 to 90 nm, 5 to 80 nm, 5 to 70 nm, 5 to 60 nm, 5 to 50 nm, 5 to 40 nm, 10 to 90 nm, 10 to 70 nm, 10 to 60 nm, 10 to 50 nm, 10 to 40 nm, 10 to 30 nm, 20 to 90 nm, 20 to 80 nm, 20 to 70 nm, 20 to 60 nm, 20 to 50 nm, 30 to 90 nm, 30 to 70 nm, 30 to 60 nm, 30 to 50 nm, 40 to 90 nm, 40 to 80 nm, 40 to 70 nm and 40 to 60 nm.

When the size of the ceria nanocomposite is within the above range, the dispersibility is good, the reactivity is maintained, and the biocompatibility is excellent in water and in vivo.

The ceria nanocomposite can be produced according to a known method, for example, the method described in Korean Patent Laid-Open Publication No. 2013-0114469.

Specifically, a cerium-surfactant complex is formed by reacting a cerium precursor with a surfactant in a first organic solvent, and deionized water is added while heating the cerium-surfactant complex solution to form ceria nanoparticles surrounded by a surfactant Then, the ceria nanoparticles surrounded by the surfactant in a second organic solvent may be capped with phospholipid-polyethylene glycol to prepare a ceria nanocomposite.

The first organic solvent may be selected from the group consisting of xylene, toluene, mesitylene, octyl ether, hexyl ether, decyl ether, pyridine, dimethyl sulfoxide, dimethylformamide, octane, decane, dodecane, tetradecane, hexadecane, Can be xylenes.

Wherein the cerium precursor is selected from the group consisting of cerium (III) acetate hydrate, cerium (III) acetylacetonate hydrate, cerium (III) carbonate hydrate, cerium (IV) hydroxide, cerium (III) (III) chloride, cerium (III) chloride heptahydrate, cerium (III) bromide, cerium (III) iodide, cerium (III) nitrate hexahydrate, cerium (III) oxalate hydrate, cerium Cerium (III) sulfate hydrate, cerium (IV) sulfate, and the like, preferably, cerium (III) acetate hydrate.

The heating temperature in the ceria nanoparticle forming step may be 80 ° C to 95 ° C, and the heating time may be 2 hours to 4 hours.

The second organic solvent may be chloroform, dichloromethane, pentane, hexane, heptane, cyclohexane, ethyl acetate, tetrahydrofuran, diethyl ether, trichlorethylene and the like, preferably chloroform.

The pharmaceutical composition may further comprise pharmaceutically acceptable additives, for example, pH adjusting agents, isotonic agents, preservatives, other excipients, and the like.

The administration route of the composition may be intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intracerebral administration, intracerebral administration, intrathecal administration, epidural administration, and the like, preferably intravenous administration.

The daily dosage of the ceria nanocomposite may be variously adjusted depending on the patient's age, symptoms, dosage form, and the like. For example, the daily dosage of the ceria nanocomposite may be 0.5-5 mg / kg, preferably 1-2 mg / kg.

The ceria nanocomposite as described above efficiently reaches the cytoplasm through the cell membrane to induce volume reduction in the non-traumatic intracranial hemorrhage site and bleeding site, reduction of brain volume or reduction of CD68 positive cell number, And can be used to treat non traumatic intracranial hemorrhage.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are intended to be illustrative of the present invention, but the present invention is not limited thereto.

[ Example ]

Manufacturing example  One. Celia  Manufacture of Nanocomposites

1-1. Celia  Manufacture of nanoparticles

1 mmol (0.4 g) of cerium (III) acetate hydrate (98%, Sigma-Aldrich) and 12 mmol (3.2 g) of _C18 content (about 80-90% , Acros Organics, Belgium) were added and mixed. The mixed solution was ultrasonicated at room temperature for 15 minutes and then heated to 90 ° C at a rate of 2 ° C / min. To this solution was then added 1 mL of deionized water and stirred vigorously at 90 DEG C to initiate the hydrolysis sol-gel reaction to see if the solution turned off-white. After confirming that the solution turned into a cloudy yellow colloid state, it was aged at 90 ° C for 3 hours and cooled to room temperature. The precipitated ceria nanoparticles were isolated by washing with 100 mL of acetone and centrifuging at a speed of 1500 rpm to remove organic residues that might remain. The separated ceria nanoparticles were stored in chloroform (10 mg / mL). The size of the ceria nanoparticles obtained by the above procedure was about 4 nm.

1-2. Phospholipid- With polyethylene glycol (PEG) Capped Celia  Manufacture of Nanocomposites

The ceria nanoparticles prepared in the above 1-1 were capped with a phospholipid-PEG to prepare a ceria nanocomposite.

First, 5 ml of ceria nanoparticles dispersed in chloroform was added to 1,2-distearoyl- sn -glycero-3-phosphoethanolamine- N- [methoxy (polyethylene glycol) -2000] (mPEG- Ml < / RTI > containing 10 ml of chloroform. The chloroform was then evaporated using a rotary evaporator and incubated at 70 ° C under reduced pressure for 3 hours to remove any residual chloroform that had not evaporated. An additional 5 ml of water was added to give a clear colloidal suspension. Thereafter, the suspension was filtered using a 0.4 mm filter, and the remaining mPEG-2000 PE was removed by ultracentrifugation to obtain a ceria nanocomposite in which ceria nanoparticles were capped with phospholipid-PEG. The ceria nanocomposite was dispersed in distilled water and stored. The size of the ceria nanocomposite obtained by the above procedure was about 18 nm.

[ Test Example ]

Test Example  1. Lactate dehydrogenase analysis (Lactate dehydrogenase  assay; LDH  assay)

LDH assay was performed to examine the therapeutic effect of the ceria nanocomposite prepared in Preparation Example 1.

Specifically, Raw 264. 7 cells (ATCC, Manassas, USA) were inoculated four times into 96-well plates (3 × 10 ⁴ cells / well) and cultured in medium at 37 ° C. for 24 hours. The medium containing 10% FBS used for tissue culture was suspended in DMEM medium (Dulbecco's Modified Eagle Medium, Welgene, Germany) containing 1% FBS to prevent albumin production from hemin binding 30 min before performing LDH analysis. Korea) (without phenol red). The total amount of LDH in the cells was confirmed by adding 2% Triton X-100 to dissolve the cells (positive control). In each well, 100 μM of hemine was added per medium, and then ceria nanocomposite of various concentrations (0 mM, 0.05 mM, 0.1 mM) was added and the amount of LDH released was measured for 6 hours. Significance level * p <0.05). At this time, negative control was defined as not adding any hemine and ceria nanocomposite to the cells.

As shown in FIG. 1, it can be seen that the amount of LDH released decreases when ceria nanocomposite is added together, as compared with the case where only hemin is added. Also, it can be seen that as the concentration of the ceria nanocomposite increases, the amount of LDH emission decreases remarkably. Thus, it can be seen that the ceria nanocomposite passes through the cell membrane and efficiently reaches the cytoplasm.

Test Example  2. Active Oxygen species (reactive oxygen species, ROS ) Removal Effect Evaluation - Excess Oxide Anion Analysis ( Superoxide -anion assay)

In order to investigate the effect of ceria nanocomposite on the removal of reactive oxygen species, a superoxide-anion assay was performed by the following method.

Specifically, U937 cells (Korean Cell Line Bank) cultured in 96-well plates for 48 hours were activated for 24 hours via lipopolysaccharide (LPS). Ceria nanocomposites were then prepared at various concentrations (0 mM, 0.05 mM, 0.1 mM). At this time, the ceria nanocomposite was fluorescently labeled with rhodamine-isothiocyanate (RITC). Specifically, the ceria nanocomposite was dissolved in propylene glycol methyl ether acetic acid (PMA, 20 μg / mL, Sigma-Aldrich), enhancer solution (5 μL, Sigma-Aldrich), luminol solution (5 μL, Sigma- (89 μL of the assay buffer and 100 μL of the assay medium), respectively, to obtain solutions having different concentrations of the ceria nanocomposite. After addition of the solution to the wells (5.0 x 10 ⁵ cells / well), fluorescence intensity was measured for 4 hours at 5 minute intervals. The results are shown in FIG. 2 as a graph (significance level * p < 0.05).

As shown in FIG. 2, it can be confirmed that the fluorescence emission amount decreases when the ceria nanocomposite is included. From this, it can be seen that the number of active oxygen species derived from LPS decreased.

Test Example  3. Active Oxygen species (reactive oxygen species, ROS ) Removal Effect Evaluation - Dichlorodihydrofluorescein Diacetate  analysis( Dichlorodihydrofluorescein diacetate ; DCF -DA assay)

In order to investigate the effect of ceria nanocomposite concentration on the removal of reactive oxygen species, dichlorodihydrofluorescein diacetate (DLC) was analyzed by the following method. DCF-DA assay).

Specifically, U937 cells (Korean Cell Line Bank) were stimulated with 1 μg / mL of LPS for 6 hours. Then, the cells were washed with phosphate buffered saline (PBS), washed with PBS at 37 ° C. containing 2 μM of DCF- &Lt; / RTI &gt; The cells were incubated at about 37 ° C for 20 minutes and 1 mL of PBS (or 1 mL of PBS without hydrogen peroxide) containing 100 μM hydrogen peroxide was added to absorb the hydrogen peroxide into the cells.

The cells were then incubated in the dark room (room temperature) for 15 minutes. The fluorescence intensity (fluorescence intensity) at 488 nm (excitation state) and 535 nm was measured using a flow cytometer (FACSCalibur, BD Science). The results are shown in Fig. 3 and Fig. 4 (significance level * p < 0.05).

As shown in FIG. 3, when the average fluorescence intensity was quantified, when the cells were treated with hydrogen peroxide, the number of active oxygen species in the cells increased, and in this state, the ceria nanocomposite was treated with 0.1 mM And it was confirmed that there was a reduction effect of active oxygen species. In addition, when the active oxygen species contained in the cells were measured without treatment with hydrogen peroxide, 0.1 mM of the ceria nanocomposite had an effect of inhibiting it.

In addition, as shown in FIG. 4, histograms of cells showing fluorescence in each group also show the same results as in FIG.

Test Example  4. Evaluation of anti-inflammatory effects -NO assay (NO production quantitative analysis)

In order to evaluate the anti-inflammatory effect according to the concentration of the ceria nanocomposite, the release amount of NO ₂ ^- was measured using a grease reagent kit (Promega, Madison, USA) as follows.

Specifically, Raw 264.7 cells were treated at a concentration of 5 × 10 ⁵ cells / well in 96-well plates and injected with 1 μg / ml LPS or 1 μg / ml LPS and ceria nanocomposite at various concentrations (0.05 mM, 0.1 mM) Respectively. Thereafter, the grease reagent was added in the same volume as the supernatant of the medium. After incubation for 10 minutes at room temperature, the absorbance at 550 nm (the amount of NO ₂ ^- released) was measured. The results are shown in Figures 5 and 6 (significance level * p = 0.03, ** p = 0.03).

5 is a photograph taken after inducing a grease reaction to measure NO ₂ ^- after ceria nanocomposite is injected.

6 is a graph showing the amount of NO ₂ ^- released over time.

As shown in FIG. 5, when the ceria nanocomposite is injected together with the LPS alone (dark purple color), the color is lightened (light purple). From this, it can be seen that the ceria nanocomposite inhibits the release of NO ₂ ^- .

6, when the LPS alone was injected, the amount of NO ₂ ^- emission was significantly higher than that of the control. However, when the ceria nanocomposite was injected together, the amount of NO ₂ ^- emission decreased remarkably. Inflammatory effect.

Test Example  5. Evaluation of anti- Western Blot  Western blot analysis

In order to evaluate the anti-inflammatory effect according to the concentration of the ceria nanocomposite, Western blot analysis was performed by the following method.

Specifically, 100 μM of hemine was injected into Raw 264.7 cells under the condition of 37 ° C., 5% carbon dioxide (CO ₂ ), dark room, or 100 μM of hemin containing various concentrations of ceria nanocomposite (0.05 mM, 0.1 mM) Cells were stimulated. At this time, the ceria nanocomposite was dispersed in DMEM containing 1% FBS. The cells were scraped and then washed with PBS at 4 ° C and homogenized with a radioimmunoprecipitation assay (RIPA) buffer solution. Western blot analysis was then performed using antibodies against cyclooxygenase (COX-2, Abcam, UK).

The results are shown in Figs. FIG. 7 is a graph showing quantitative analysis of expression of COX-2 on actin expression relative to relative optical density. FIG. 8 is a graph showing the expression of COX-2 and actin according to the concentration of ceria nanocomposite by Western blot ).

7, when the cells were stimulated with hematin alone, inflammation was induced and the expression of COX-2 was elevated. When the hematin and ceria nanocomposite were co-injected, the expression of COX-2 . Further, it can be seen that as the concentration of the ceria nanocomposite is increased, the expression rate of COX-2 is remarkably decreased.

8 shows that COX-2 bands are more lighter than hemin-only injections of hemin and ceria nanocomposites, and COX-2 expression is significantly reduced.

Test Example  6. Cell death rate  evaluation- TUNEL  assay

In order to evaluate the cell death rate according to the concentration of ceria nanocomposite, TUNEL assay was performed by the following method.

Specifically, the terminal-deoxynucleotidyltransferase dUTD nick and the labeled TUNEL assay kit (Roche Appiled Science, Penzberg, Germany) were used to measure the mortality of Raw 264.7 cells stimulated in the same manner as in Test Example 5. [ Were used.

First, Raw 264.7 cells were fixed with 4% p-formaldehyde (100 uL) and osmotic treated with osmotic solution (0.1% sodium citrate containing 0.1% Triton X-100) (100 uL) for about 2 minutes at 5 [ . Next, 50 μL of the TUNEL reaction mixture was stained with 4,6-diamidino-2-phenylindine-stone (DAPI). The sample was observed under a fluorescence microscope (Leica DM5500B; Leica Biosystems Inc., USA) and the results are shown in FIG. FIG. 9 shows DAPI staining (blue) images, TUNEL-positive cells (dead cells, green) images, and merged images thereof. In the composite image, dead cells are indicated by arrows. In addition, quantitative analysis was performed and shown in FIG. 10 (significance level * p < 0.05).

9, when the ceria nanocomposite was injected, the number of TUNEL-positive cells (dead cells) was remarkably decreased as compared to the case of injecting hemin alone. As the concentration of the ceria nanocomposite increased, The number of cells can be seen to decrease. In addition, as shown in FIG. 10, it can be seen that when the ceria nanocomposite is injected, the number of apoptotic cells is decreased as compared with the case of injecting hemin alone. Also, it can be seen that as the concentration of the ceria nanocomposite increases, the number of dead cells decreases.

Test Example  7. Evaluation of anti-inflammatory effect - hematoma ( hematoma ) Volume measurement

To evaluate the anti-inflammatory effect of the ceria nanocomposite, SD rats (Coatech Co., Ltd.) were anesthetized with isoflurane and then 1 μL of physiological saline containing 0.5 unit of collagenase VII (Sigma-Aldrich) Intracranial hemorrhage (ICH) was induced by intramuscular injection into the rat's striatum using a rat Stereotactic coordinate, carried on a Hamilton syringe (approximately Bian). At the 6th and 30th hours of ICH induction, 0.5mg / kg of ceria nanocomposite or PBS of the same volume was intravenously injected, and the brain of the rat was extracted 72 hours after induction of ICH, and the ICH- and cut in a coronal direction. And then successively cut to a thickness of 1 mm to obtain both the front and rear of the needle entry site. 11 is a schematic diagram showing the process of inducing ICH in rats, the injection timing, injection amount, and measurement timing of ceria nanocomposite. Test Examples 8 to 12 were also conducted according to the above-described schematic diagrams.

Digital photographs of the serially sectioned sections were taken and the volume of bleeding was measured using an image analysis program (Image J, National Institutes of Health, USA). Total bleeding volume (mm ³ ) was calculated by multiplying the bleeding sites in each section and then multiplying the distance between each section. The results are shown in Fig. At this time, the case of injecting PBS was compared as a control group.

The result of FIG. 12 shows that the ceria nanocomposite was injected and the hematoma volume was equal to that of the control group.

Test Example  8. Evaluation of anti- Brain quantity  Measure

With respect to the rats of Test Example 7, when the damage degree of the brain was the maximum, the brain was divided into two hemispheres along the center line, and the brain stem and the cerebellum were removed. The two divided hemispheres were weighed immediately using an electronic analytical balance (HS320S microbalance, Hansung) to obtain the wet weight. And then dried at 100 DEG C for 24 hours to measure the dry weight. The wet weight and the dry weight were expressed as a percentage using the following equation (1). The results are shown graphically in Fig. At this time, the case of injecting PBS was compared as a control group.

[Equation 1]

As shown in FIG. 13, when the ceria nanocomposite was injected, the brain volume was reduced by about 1.14% as compared with the control group. From the result, it can be expected that the ceria nanocomposite is effective for treatment of intracranial hemorrhage have.

Test Example  9. Evaluation of anti- CD68  Cell quantitative analysis

The rats of Test Example 7 were sequentially perfused with PBS and p-formaldehyde (PFA). After soaking in 4% PFA for 24 hours, the brains of rats were frozen in PBS containing 30% sucrose. Then, a coronal section of 40 mu m was obtained at -20 DEG C using a cryostate microtome (Leica).

Subsequently, nonspecific binding of the antibody was blocked using PBS containing 10% normal serum, and CD68 (ab125212, Abcam) labeled with microglial cells and macrophages was treated with the primary antibody and incubated at 4 ° C for 16 hours Lt; / RTI &gt; The nucleus was stained with 4,6-diaminodino-2-phenylindole. Each section was washed with PBS and incubated at room temperature for 2 hours to incubate the secondary antibody. Alexa Fluor 488 goat anti-rabbit IgG antibody (A11008, Invitrogen) was used as a secondary antibody. The stained cells were observed with an immunofluorescence microscope (Leica DM5500B; Leica Biosystems Inc., USA). The results are shown in Fig.

The stained cells were quantitatively analyzed using NIH ImageJ software (ImageJ 1.50c4; National Institutes of Health, USA). At this time, an analysis was performed on the entire coronal brain slice adjacent to the center of the needle track (depth 1 mm). The results are shown in FIG. 15 (significance level * p < 0.05).

As shown in FIG. 14, when the ceria nanocomposite was injected, it could be seen that the number of CD68-positive cells was observed around the bleeding hemorrhage than when the PBS was injected. The same result can be confirmed also in the result of Fig.

Test Example  10. Evaluation of Anti-inflammatory Effect - Western Blot  Western blot analysis

Western blot analysis was carried out in the following manner to evaluate the anti-inflammatory effect according to the concentration of the ceria nanocomposite.

Specifically, the brain of the rat of Test Example 7 was pulverized to obtain a cerebral hemispherical pulverization solution. Subsequently, 50 mg of the protein was quantified and Western blot analysis was performed using an antibody against cyclooxygenase (COX-2, Abcam, UK). The results are shown in Figs.

16, the expression of COX-2 was elevated due to the inflammation in the intracranial hemorrhage-induced rat, and the expression of COX-2 was decreased when the ceria nanocomposite was injected .

The results of FIG. 17 show that COX-2 protein bands were more lighter than those not injected with ceria nanocomposite, and the expression of COX-2 significantly decreased.

From this, it can be seen that the ceria nanocomposite has an effect of inhibiting inflammation caused by intracranial hemorrhage.

Test Example  11. Celia  Evaluation of distribution of nanocomposites

In order to evaluate the distribution of ceria nanocomposite in the brain in which intracranial hemorrhage occurred, cital nanocomposite labeled with RITC at 6 hours after intracranial hemorrhage induction was injected into the brain of rats of Test Example 7. After that, the brain was extracted 24 hours after intracranial hemorrhage induction, and a slide for fluorescence microscopy was prepared and observed using a fluorescence microscope (Leica DM5500B; Leica Biosystems Inc., USA). The results are shown in Fig.

As shown in FIG. 18 (a), the RITC-labeled ceria nanocomposite is dispersed mainly through the extracellular space and can not pass through the ventricles or gray matter. Furthermore, as shown in (b) and (c), it can be seen that the RITC-labeled ceria nanocomposite is mainly distributed in the extracellular space but is absorbed into the cell. In addition, as shown in (d), (e) and (f), when the entire brain tissue slide was observed with a fluorescence microscope, the RITC-labeled ceria nanocomposite was mainly distributed around the hematoma, But not in the opposite hemisphere.

Test Example  12. Through 3D reconstruction Celia  Evaluation of nanocomposite distribution

In order to evaluate the distribution of ceria nanocomposite in the brain in which intracranial hemorrhage occurred, the following test was performed using the brain of the rat of Test Example 11. Specifically, after adding an X100 high-power field image with 561 nm light, a raw image of the entire brain slice was created. Then the raw image ^{MATLAB ® (MATLAB ® R2015b 8.6.0.267246,} 64-bit, MathWorks, Inc., USA) was used to, true color (truecolor) image of 4766 X 6298 pixels of red (R), green ( (0.2989 * R + 0.5870 * G + 0.1140 * B) by weighting with gray (G) and blue (B).

To generate a two-dimensional histogram, the image was divided into squares (200 x 280 pixels) and the intensity values of the average grayscales of each fraction were calculated taking into account the shape of the tissue section. Relative fluorescence intensity refers to the average grayscale intensity versus grayscale intensity. The calculated (x, y) data values were obtained by calculating the kernel density (standard deviation 100) using a Gaussian low-pass filter of size [20 20]. A three-dimensional plot shape was created using the obtained x, y and density values. The results are shown in Figs. 19 and 20.

As shown in FIGS. 19 and 20, it can be seen that the ceria nanocomposite is distributed widely in the hemispherical hemisphere.

Claims (9)

A ceria nanocomposite comprising a ceria nanoparticle, a surfactant and a phospholipid-polyethylene glycol (PEG)
Wherein said ceria nanoparticles are surrounded by a surfactant and are capped with a phospholipid-polyethylene glycol.
delete The method according to claim 1,
C _6-20 alkylamines wherein the surfactant is oleylamine, octylamine, hexadecylamine or octadecylamine; Alkyltrimethylammonium salts such as cetyltrimethylammonium bromide, octyltrimethylammonium bromide or dodecyltrimethylammonium bromide; (Ethylene oxide) ₂₀ (propylene oxide) ₇₀ (ethylene oxide) ₂₀ poly (alkylene oxide) tri-co-polymer; And an alkali salt of oleic acid, linoleic acid, lauric acid, myristic acid, palmitic acid or C _12-18 fatty acid which is a stearic acid, for the treatment of non traumatic intracerebral hemorrhage.
The method according to claim 1,
Wherein the ceria nanocomposite has a size of 5 to 100 nm.
5. The ceria nanocomposite material according to claim 4,
Reacting a cerium precursor with a surfactant in a first organic solvent to form a cerium-surfactant complex;
Adding deionized water while heating the cerium-surfactant complex solution to form ceria nanoparticles surrounded by the surfactant; And
Characterized in that it is prepared by capping ceria nanoparticles surrounded by said surfactant in a second organic solvent with phospholipid-polyethylene glycol.
6. The method of claim 5,
Wherein the first organic solvent is selected from the group consisting of xylene, toluene, mesitylene, octyl ether, hexyl ether, decyl ether, pyridine, dimethyl sulfoxide, dimethyl formamide, octane, decane, dodecane, tetradecane and hexadecane &Lt; / RTI &gt; wherein the pharmaceutical composition is for the treatment of non-traumatic intracranial hemorrhage.
6. The method of claim 5,
Wherein the cerium precursor is selected from the group consisting of cerium (III) acetate hydrate, cerium (III) acetylacetonate hydrate, cerium (III) carbonate hydrate, cerium (IV) hydroxide, cerium (III) (III) chloride, cerium (III) chloride heptahydrate, cerium (III) bromide, cerium (III) iodide, cerium (III) nitrate hexahydrate, cerium (III) oxalate hydrate, cerium Cerium (III) sulfate hydrate, and cerium (IV) sulfate.
6. The method of claim 5,
Wherein the second organic solvent is selected from the group consisting of chloroform, dichloromethane, pentane, hexane, heptane, cyclohexane, ethyl acetate, tetrahydrofuran, diethyl ether and trichlorethylene. A pharmaceutical composition.
9. The method according to any one of claims 1 to 8,
Wherein said composition induces a decrease in hematoma volume, brain volume reduction, or CD68 positive cell number in the non-traumatic intracranial hemorrhage site and the bleeding site.

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