KR102284707B1 - Nanoparticles structure - Google Patents

Abstract

A nanoparticulate structure is provided. The nanoparticle structure includes a core including a cluster to which magnetic nanoparticles are assembled, and a shell positioned on the surface of the core and including ceria nanoparticles. Any one of the magnetic nanoparticles is in contact with at least one other magnetic particle among the magnetic nanoparticles, and the ceria nanoparticles are in contact with the core.

Description

Nanoparticle structure {NANOPARTICLES STRUCTURE}

The present invention relates to nanoparticulate structures.

The neuropathological role of amyloid-β in Alzheimer's disease (AD) has become a major focus of Alzheimer's disease research since amyloid-β plaques were first observed in the postmortem brain of Alzheimer's disease patients. Amyloid-β accumulation in the brain leads to the formation of senile plaques associated with neuronal dysfunction and death, and elevation of amyloid-β peptide has been considered as a major cause of the pathogenesis of Alzheimer's disease. Thus, certain amyloid-β antibodies are administered or generated into the bloodstream of Alzheimer's disease patients to act as peripheral amyloid-β sinks or to activate microglial phagocytosis of amyloid-β plaques in the brain by immunization generated. Extensive research has been done to reduce β deposits. However, the previous amyloid-β immunotherapy has problems such as inducing unwanted side effects such as meningitis and micro-bleeding, so the development of clinically relevant technology for reducing amyloid-β in patients with Alzheimer's disease has not progressed.

In order to solve the above problems, the present invention provides a novel nanoparticle structure.

The present invention provides a nanoparticulate structure that can be used to treat diseases without side effects.

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

Nanoparticle structures according to embodiments of the present invention include a core including a cluster to which magnetic nanoparticles are assembled, and a shell positioned on the surface of the core and including ceria nanoparticles. Any one of the magnetic nanoparticles is in contact with at least one other magnetic particle among the magnetic nanoparticles, and the ceria nanoparticles are in contact with the core.

The magnetic nanoparticles may include iron oxide nanoparticles.

The nanoparticle structure may further include an antibody that binds to the ceria nanoparticles. The antibody may comprise an amyloid-β antibody. The antibody may be bound to the ceria nanoparticles by polyacrylic acid.

The nanoparticle structure may further include a dispersible compound bonded to the ceria nanoparticles, and the dispersible compound may provide dispersibility to the nanoparticle structure. The dispersible compound may include PEG.

The nanoparticle structure may have a hydrodynamic diameter of 200 to 400 nm.

The shell may be a single layer of the ceria nanoparticles.

The nanoparticle structure and the blood cleansing system using the same according to embodiments of the present invention can be easily used to treat various diseases such as Alzheimer's disease. The nanoparticulate structure can specifically capture amyloid-β peptide from blood with high capture efficiency, and can be easily recovered from blood by magnetic separation. The nanoparticle structure may be injected into the blood from outside the body by the blood cleansing system to perform blood cleansing, but is not injected into the body. The nanoparticulate structure and the blood cleansing system do not cause side effects such as oxidative stress, infection, and cardiovascular disease, and WBC, RBC, PLT, NEU, MCV, and MPV values do not change significantly, which is advantageous for the patient. In addition, during blood cleansing, a large amount of various types of reactive oxygen species can be removed, thereby relieving oxidative stress and preventing inflammation.

1 shows an iron oxide/ceria nanoparticle structure according to an embodiment of the present invention.
FIG. 2 schematically shows the formation process of the iron oxide/ceria nanoparticle structure of FIG. 1 .
3 is a view for explaining the versatility of the iron oxide / ceria nanoparticle structure of FIG.
4 shows a TEM image of iron oxide nanoparticles.
5 shows a TEM image of an iron oxide nanoparticle cluster.
6 shows a TEM image of ceria nanoparticles.
7 shows a TEM image of an iron oxide/ceria nanoparticle structure.
8 shows a magnetization curve of an iron oxide/ceria nanoparticle structure measured at a temperature of 300K.
9 shows the SOD-like activity of the iron oxide/ceria nanoparticle structure according to the ceria concentration compared to that of the ceria nanoparticles.
Figure 10 shows the CAT-like activity of the iron oxide / ceria nanoparticle structure according to the ceria concentration compared to that of the ceria nanoparticles.
11 shows a blood cleansing system according to an embodiment of the present invention.
12 shows changes in plasma amyloid-β before and after blood cleansing treatment using iron oxide/ceria nanoparticle structures.
13 shows the level of reactive oxygen species in plasma after blood cleansing treatment using iron oxide/ceria nanoparticle structures.
14 shows the concentration ratio of amyloid-β to GAPDH in mouse brain after blood cleansing treatment using iron oxide/ceria nanoparticle structures.
15 shows amyloid-β plaque levels in mouse brain after blood cleansing treatment using iron oxide/ceria nanoparticle structures.
16 shows the expression level of GFAP in mouse brain after blood cleansing treatment using iron oxide/ceria nanoparticle structures.
17 is a confocal laser scanning microscopy (CLSM) image showing the expression of amyloid-β of FIG. 15 and GFAP of FIG. 16 .
18 to 23 show WBC, RBC, PLT, NEW, MCV, and MPV in mouse blood after blood cleansing treatment using iron oxide/ceria nanoparticle structures, respectively.

Hereinafter, the present invention will be described in detail through examples. Objects, features and advantages of the present invention will be easily understood through the following examples. The present invention is not limited to the embodiments described herein, and may be embodied in other forms. The embodiments introduced here are provided so that the disclosed content can be thorough and complete, and so that the spirit of the present invention can be sufficiently conveyed to those of ordinary skill in the art to which the present invention pertains. Therefore, the present invention should not be limited by the following examples.

Although terms such as first, second, etc. are used herein to describe various elements, the elements should not be limited by these terms. These terms are only used to distinguish the elements from each other. Also, when an element is referred to as being coupled to another element, it may be directly coupled to the other element or a third element may be interposed therebetween.

In the drawings, the size of the elements, or the relative sizes between the elements, may be exaggerated for a clearer understanding of the present invention. In addition, the shape of the element shown in the drawings may be slightly changed due to variations in the manufacturing process, or the like. Accordingly, the embodiments disclosed in the present specification should not be limited to the shapes shown in the drawings unless otherwise noted, but should be understood to include some degree of deformation.

The term A/B nanoparticle structure as used herein is a nanoparticle having a core (A)-shell (B) structure in which a plurality of B nanoparticles are disposed on the surface of a cluster in which A nanoparticles or a plurality of A nanoparticles are assembled. means structure. For example, the iron oxide/ceria nanoparticle structure refers to a nanoparticle structure in which a cluster in which a plurality of iron oxide nanoparticles is assembled is a core, and a plurality of ceria nanoparticles disposed on the surface of the cluster is a shell. The B (shell) may partially cover the A (core) surface or cover the entire surface.

Nanoparticle structures according to embodiments of the present invention, a core including a first nanoparticle, a shell positioned on the surface of the core and including a second nanoparticle, a dispersible compound bound to the second nanoparticle, and an antibody may include.

The first nanoparticles may include magnetic nanoparticles. The first nanoparticles may include first metal oxide nanoparticles. The first metal oxide nanoparticles may include iron oxide nanoparticles. The core may include a cluster in which a plurality of the first nanoparticles are assembled.

The second nanoparticles may include catalytic nanoparticles. The second nanoparticles may include second metal oxide nanoparticles. The second metal oxide nanoparticles may include ceria nanoparticles.

The antibody and the dispersible compound may be bound to the second metal oxide nanoparticles by polyacrylic acid. The antibody may comprise an amyloid-β antibody. The dispersible compound may include PEG.

The nanoparticle structure may have a hydrodynamic diameter of 200 to 400 nm.

1 shows an iron oxide/ceria nanoparticle structure according to an embodiment of the present invention.

Referring to FIG. 1 , the iron oxide/ceria nanoparticle structure 10 may include a core 110 , a shell 120 , a dispersible compound 130 , and an antibody 140 .

The core 110 may include iron oxide nanoparticles 111 . For example, the core 110 may include a cluster in which a plurality of iron oxide nanoparticles 111 are assembled. The core 110 may have magnetism, whereby the iron oxide/ceria nanoparticle structure 10 may be separated from the blood after the blood cleansing treatment. The iron oxide nanoparticles 111 may have a diameter of about 10 nm, and the core 110 may have a diameter of about 200 nm.

The shell 120 may include ceria nanoparticles 121 positioned on the surface of the core 110 . In addition, the shell 120 may be formed of a single layer of ceria nanoparticles 121 . The ceria nanoparticles 121 may remove reactive oxygen species during the blood cleansing process. The ceria nanoparticles 121 may have a diameter of about 3 nm.

The dispersible compound 130 may be bound to the ceria nanoparticles 121 to provide dispersibility to the iron oxide/ceria nanoparticle structure 100 . The dispersible compound 130 may have water dispersibility and/or oil dispersibility. The dispersible compound 130 may include, for example, polyethylene glycol (PEG) or Lipid-PEG. The dispersible compound 130 may be bound to the ceria nanoparticles 121 by polyacrylic acid.

The antibody 140 is bound to the ceria nanoparticles 121 and can be used for various blood cleansing. For example, antibody 140 may include an amyloid-β antibody and may be used to treat Alzheimer's disease. The antibody 140 may be bound to the ceria nanoparticles 121 by polyacrylic acid.

FIG. 2 schematically shows the formation process of the iron oxide/ceria nanoparticle structure of FIG. 1 .

Referring to FIG. 2 , iron oxide nanoparticles 111 are formed. The iron oxide nanoparticles 111 may be synthesized by thermal decomposition of an iron-oleate complex. Iron(III) chloride (10.8 g, Aldrich, 97%) and sodium oleate (36.5 g, TCI, 97%) are dissolved in a mixture of 80 ml ethanol, 60 ml deionized water, and 140 ml hexane. This mixed solution was reacted at 60° C. for 8 hours and then cooled to room temperature. The upper organic layer was separated and washed three times with deionized water. The iron-oleate complex can be obtained by evaporating hexane from the separated organic solution. A mixed solution of iron-oleate (1.8 g), oleic acid (0.28 g, Aldrich, 90%), and 1-octadecene (12 g, Aldrich, 90%) was heated to 320 °C (1 °C/min). speed) is heated. After aging for 30 minutes with vigorous stirring at this temperature, the mixed solution is cooled to room temperature. Accordingly, the iron oxide nanoparticles 111 having a size of about 10 nm are formed. The iron oxide nanoparticles 111 are washed with an excess of ethanol and purified by centrifugation. After repeating the washing and centrifugation steps two more times, the obtained iron oxide nanoparticles 111 are dispersed in chloroform.

The ceria nanoparticles 121 are formed. A mixed solution of cerium(III) acetate (0.32 g, Aldrich, 99.9%), oleylamine (3.2 g, acros, 85%), and xylene (13 g) is vigorously stirred at room temperature for 12 hours. The mixed solution is heated at a temperature increase rate of 2°C/min. Deionized water (1 g) is rapidly injected into the mixed solution at 90°C. The mixed solution is maintained at this temperature for 3 hours and then cooled to room temperature. Accordingly, ceria nanoparticles 121 having a size of about 3 nm are formed. After washing the solution with an excess of ethanol, the ceria nanoparticles 121 are separated by centrifugation. After repeating the washing and centrifugation steps two more times, the obtained ceria nanoparticles 121 are dispersed in chloroform.

The iron oxide/ceria nanoparticle structure 100 is formed by coating the ceria nanoparticles 121 on the self-assembled cluster 110 of the iron oxide nanoparticles 111 . About 200 nm of assembled iron oxide nanoparticles (111) by mixing iron oxide nanoparticles (150 mg) in chloroform (4.5 g) with dodecyltrimethylammonium bromide (150 mg, Aldrich, 98%) in deionized water (10 g) with vigorous stirring An iron oxide nanoparticle cluster 110 of the size is formed. The chloroform is evaporated and the solution is mixed with polyacrylic acid (0.9 g, Aldrich) in ethylene glycol (11.1 g, Aldrich, 99.8%). After washing with excess deionized water, the iron oxide nanoparticle cluster 110 is separated by centrifugation. In order to coat the ceria nanoparticles 121 on the iron oxide nanoparticle clusters 110, the iron oxide nanoparticle clusters 110 are mixed with the ceria nanoparticles 121 in separately prepared chloroform. After stirring overnight, the iron oxide/ceria nanoparticle structure 100 is separated by centrifugation. The iron oxide/ceria nanoparticle structure 100 is dispersed in deionized water and mixed with polyacrylic acid (0.9 g), and after stirring for 1 hour, excess polyacrylic acid is removed by centrifugation.

The antibody 140 is included by being bound to the iron oxide/ceria nanoparticle structure 100 through a covalent bond between polyacrylic acid and the antibody. 1-ethyl-3-(3-dimethylaminopropyl)urea hydrochloride (1 mg, Aldrich, 99%) and N-hydroxysuccinic in 2-(N-morpholino)ethanesulfonic acid buffer (100 μL, pH 4.7) Mead (1 mg, Aldrich, 98%) was added to the iron oxide/ceria nanoparticle structure 100 (1 mg [Fe]) dispersed in deionized water (1 mL) and maintained at constant temperature for 30 minutes. Then, the iron oxide/ceria nanoparticle structure 100 is separated by centrifugation and mixed with anti-human amyloid-β antibody (500 μL, BioLegend, 800702).

After 1 hour, the antibody-bound iron oxide/ceria nanoparticle structure 100 is separated by centrifugation and dispersed in a borate buffer. PEG(2000)-amine (50 mg) in phosphate buffer (PBS) was added to the solution, and maintained at constant temperature for 2 hours, so that PEG 130 was bound to the iron oxide/ceria nanoparticle structure 100 and contained therein. The iron oxide/ceria nanoparticle structure 100 is separated by centrifugation after washing, dispersed in phosphate buffer (500 μL), and stored at 4° C. before use.

3 is a view for explaining the versatility of the iron oxide / ceria nanoparticle structure of FIG.

Referring to FIG. 3 , the iron oxide nanoparticles 111 assembled in the core 110 of the iron oxide/ceria nanoparticle structure 100 are amyloid-β captured by the antibody 140 by generating a large magnetic adsorption force toward the external magnet. Allows the separation of peptides (Aβ). In the shell 120 of the iron oxide / ceria nanoparticle structure 100, the ceria nanoparticles 121 are regenerative catalysts that remove reactive oxygen species (ROS) generated by the immune reaction during the reaction (blood cleansing) process. it works Blood amyloid-β cleansing treatment for 5XFAD transgenic mice reduces the amount of amyloid-β plaques in the brain, making it possible to prevent and treat Alzheimer's disease.

4 shows a TEM image of iron oxide nanoparticles, FIG. 5 shows a TEM image of an iron oxide nanoparticle cluster, FIG. 6 shows a TEM image of ceria nanoparticles, and FIG. 7 is a TEM image of an iron oxide/ceria nanoparticle structure. indicates.

4 to 7 , iron oxide nanoparticles having a size of about 10 nm are synthesized by thermal decomposition of an iron oleate complex ( FIG. 4 ). Transmission electron microscopy (TEM) images show the super-superlattice structure of self-assembly due to the size uniformity of the iron oxide nanoparticles (Fig. 5). 6 shows ceria nanoparticles having a size of about 3 nm, and the ceria nanoparticles are coated on iron oxide nanoparticle clusters (FIG. 7). The thickness of the ceria nanoparticle layer is about 3 nm, which means that a single layer of ceria nanoparticles covers the iron oxide nanoparticle clusters.

The core/shell structural assembly of the iron oxide/ceria nanoparticulate structure is coated with polyacrylic acid for structural stabilization and for covalent bonding of amyloid-β antibody with anti-fouling polyethylene glycol (PEG). The formed iron oxide/ceria nanoparticle structure has a hydrodynamic diameter of about 250 nm and a ζ-potential value of -45 mV, and the hydrodynamic diameter and ζ-potential value increase to about 330 nm and -23 mV, respectively, after binding of the antibody and PEG.

Antibodies of the iron oxide/ceria nanoparticle structure can be identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and are stably maintained in the iron oxide/ceria nanoparticle structure without noticeable decrease in activity for at least 1 month.

8 shows a magnetization curve of an iron oxide/ceria nanoparticle structure measured at a temperature of 300K.

Referring to FIG. 8 , the magnetization curve of the iron oxide/ceria nanoparticulate structure measured at room temperature does not show any hysteresis loop expected from a superparamagnetic material.

9 shows the SOD-like activity of the iron oxide / ceria nanoparticle structure according to the ceria concentration compared to the ceria nanoparticles, and Figure 10 is the CAT-like activity of the iron oxide / ceria nanoparticle structure according to the ceria concentration compared to the ceria nanoparticles. indicates.

9 and 10 , the reactive oxygen species scavenging activity of iron oxide/ceria nanoparticle structures (ICSNPs) was evaluated using superoxide dismutase (SOD) and catalase (CAT) activity assays. The dose-dependent activity observed in both assays demonstrated the ability of iron oxide/ceria nanoparticle structures to remove ^{peroxide (˙O 2} ) and hydrogen peroxide (H ₂ O ₂ ) using the SOD and CAT-like activities of ceria nanoparticle shells, respectively. show clearly The low reactive oxygen species scavenging ability of the iron oxide/ceria nanoparticle structures compared to the individually dispersed ceria nanoparticles can be attributed to the low surface-to-volume ratio.

Although not shown in the figure, in the MTT assay performed to evaluate the cytotoxicity of the iron oxide/ceria nanoparticle construct on HeLa cells, the cell viability was maintained high at a high concentration of the iron oxide/ceria nanoparticle construct of 1.0 mM [Fe]. appeared to be As such, the iron oxide/ceria nanoparticle structure may have excellent biocompatibility by PEG coating, and cellular uptake of the iron oxide/ceria nanoparticle structure may be prevented due to its large size.

11 shows a blood cleansing system according to an embodiment of the present invention.

Referring to FIG. 11 , the blood cleansing system 1 includes a blood circulation unit 10 , a nanoparticle structure supply unit 20 , and a nanoparticle structure recovery unit 30 .

The blood circulation unit 10 may include a blood circulation pump 11 , a blood discharge tube 12 , and a blood injection tube 13 . The blood circulation pump 11 is disposed between the blood discharge tube 12 and the blood injection tube 13 to discharge blood out of the body and then inject it back into the body to circulate the blood. The blood circulation pump 11 may include, for example, a peristaltic pump. The blood discharge pipe 12 is disposed between the blood discharge point of the blood cleansing treatment target and the blood circulation pump 11 to discharge body blood to the outside of the body. The blood injection tube 13 is disposed between the blood injection point of the blood cleansing treatment target and the blood circulation pump 11 to inject the blood cleansing treatment into the body.

The nanoparticle structure supply unit 20 is connected to the blood circulation unit 10 to supply the nanoparticle structure 100 to the blood discharged outside the body. The nanoparticulate structure supply unit 20 may include, for example, a syringe pump. The nanoparticulate structure supply unit 20 may supply the nanoparticulate structure 100 to the blood discharge pipe 12 adjacent to the blood discharge point.

The nanoparticulate structure recovery unit 30 is connected to the blood circulation unit 10 to recover the nanoparticulate structure 100 in which the amyloid-β peptide (Aβ) is captured in blood cleansing. The nanoparticle structure recovery unit 30 may include, for example, a permanent magnet. The nanoparticle structure supply unit 30 may recover the nanoparticle structure 100 from the blood injection tube 13 adjacent to the blood injection point.

11 schematically shows an example of blood amyloid-β cleansing treatment using iron oxide/ceria nanoparticle constructs (ICSNPs) in a 5XFAD transgenic mouse as an Alzheimer's disease model.

Referring back to FIG. 11 , a peristaltic pump discharges blood from the femoral vein of anesthetized mice and circulates extracorporeally at a flow rate of 150 μL/min. An iron oxide/ceria nanoparticulate structure solution (1.8 mM [Fe]) near the starting point of the extracorporeal circulation circuit through a micromixer is injected by a syringe pump operating at a flow rate of 10 μL/min. The iron oxide/ceria nanoparticle structure is diluted in solution to an appropriate concentration in consideration of the amyloid-β peptide capture efficiency in a concentration range that does not have cytotoxicity in the MTT assay result. The blood discharged from the body is mixed with the iron oxide/ceria nanoparticle structure to which the amyloid-β antibody is bound in the extracorporeal circulation circuit, and the amyloid-β antibody specifically captures the amyloid-β peptide in the blood.

A permanent magnet is placed near the end of the circuit to separate the iron oxide/ceria nanoparticle structure and its bound amyloid-β peptide from the blood. The core of the iron oxide/ceria nanoparticle structure is composed of a number of self-assembled superparamagnetic iron oxide nanoparticles, which can be magnetically separated together with the entrapped amyloid-β peptide by applying an external magnetic field. . The ceria nanoparticles in the shell of the iron oxide/ceria nanoparticle structure can remove reactive oxygen species (ROS) that can be generated when blood meets foreign substances.

The separated iron oxide/ceria nanoparticle structure remains in the pipe without blocking the flow channel and is recovered. The treated blood is injected into the jugular vein of the mouse after magnetic separation and returned to the bloodstream.

Since the amyloid-β peptide-antibody complex and the remaining unused amyloid-β antibody are not injected into the body of the mouse, there is no need for subsequent treatment therewith.

12 shows changes in plasma amyloid-β before and after blood cleansing treatment using iron oxide/ceria nanoparticle constructs (ICSNPs).

Referring to FIG. 12 , blood amyloid-β cleansing performance is evaluated by comparing amyloid-β peptide concentrations measured before and after cleansing treatment. A total of 20 plasma samples before and after sham treatment or iron oxide/ceria nanoparticle construct treatment (5 mice per group) were obtained from 2 month old 5XFAD transgenic mice, and their amyloid-β levels were analyzed. It appears that 76% of amyloid-β peptide in blood is removed on average by the iron oxide/ceria nanoparticle structure treatment. On the other hand, the Shen group without iron oxide/ceria nanoparticle structures during blood treatment showed no significant difference between plasma amyloid-β levels measured before and after treatment. Although not shown in the figure, Western blot data also showed a decrease in plasma amyloid-β levels after cleansing treatment.

13 shows the level of reactive oxygen species in plasma after blood cleansing treatment using iron oxide/ceria nanoparticle structures (ICSNPs).

Referring to FIG. 13 , the ceria nanoparticle shell of the iron oxide/ceria nanoparticle structure helps to suppress the generation of reactive oxygen species during the blood cleansing process. In blood cleansing, if the nanoparticle structure of the present invention is not used or supernanoparticles composed only of iron oxide nanoparticles are used, the plasma reactive oxygen species level is significantly increased. An increase in the level of reactive oxygen species can be minimized by using an iron oxide/ceria nanoparticulate structure.

The effect of blood amyloid-β cleansing treatment on the amount of amyloid-β in the brain was investigated using immunostaining of brain sections. Two month old 5XFAD transgenic mice were divided into 3 groups (5 mice per group). The first group (untreated group, Tg) did not receive blood cleansing treatment. The second group (Treatment group) underwent two blood cleansing treatments at 1-month intervals using iron oxide/ceria nanoparticle structures. The last group (Sham's group, Sham) also received two treatments but did not use iron oxide/ceria nanoparticles. All mice were sacrificed at 4 months and brains were isolated and analyzed. Although different from the previously described plasma amyloid-β level comparison experiments in which each set of samples before and after blood cleansing treatment were obtained from the same mouse because each brain sample was obtained from a different mouse, a general trend can still be observed in the data. .

14 shows the concentration ratio of amyloid-β to GAPDH in mouse brain after blood cleansing treatment using iron oxide/ceria nanoparticle structures, and FIG. 15 is amyloid-β plaques in mouse brain after blood cleansing treatment using iron oxide/ceria nanoparticle structures. 16 shows the expression level of GFAP in the mouse brain after blood cleansing treatment using iron oxide/ceria nanoparticle structures, and FIG. 17 is a confocal laser showing the expression of amyloid-β of FIG. 15 and GFAP of FIG. 16 This is a confocal laser scanning microscopy (CLSM) image.

Referring to FIG. 14 , the brain amyloid-β levels of the treated group were significantly lower than the brain amyloid-β levels of the untreated group. Conversely, the brain amyloid-β levels of the Shen group did not show a significant difference from the brain amyloid-β levels of the untreated group.

Referring to FIG. 15 , the immunohistologic fluorescence analysis results of coronal sections of mouse brains (5 mice per group), similar to the immunoassay results, the treated group showed a higher level of amyloid-β plaques in the cerebral cortex compared to the untreated group. On the other hand, there was no significant difference in the Sheng group.

Referring to FIG. 16 , the expression of glial fibrillary acidic protein (GFAP) in brain astrocytes related to neuroinflammation also showed a significant decrease in the treatment group.

Referring to FIG. 17 , the results of FIGS. 14 to 16 can also be confirmed in the CLSM image.

18 to 23 show WBC, RBC, PLT, NEW, MCV, and MPV in mouse blood after blood cleansing treatment using iron oxide/ceria nanoparticle structures, respectively. Blood samples from sacrificed mice were analyzed to assess compositional changes.

18 to 20 , a complete blood count (CBC) result shows that the white blood cell (WBC), red blood cell (RBC) and platelet (PLT) concentrations of the treatment group are It shows that there is no significant difference with that of the untreated group.

21 to 23, neutrophil (NEU) concentration, mean corpuscular volumes (MCV) and mean platelet volumes, which are important parameters for evaluating the functions of WBC, RBC and PLT , MPV) also showed no significant difference between the treated and untreated groups. This indicates that blood cleansing treatment with iron oxide/ceria nanoparticulate structures did not cause serious inflammation or side effects in mice.

[Analysis Example]

SOD and CAT-like activity assays

SOD-like activity was measured using the SOD assay kit (Sigma-Aldrich, 19160). The iron oxide/ceria nanoparticle structure was mixed with 600 μL of WST-1 (water-soluble tetrazolium salt; 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfo-phenyl)-2H-tetrazolium). monosodium salt) solution at concentrations of 0, 0.06, 0.125, 0.25, 0.5 and 1 mM [Ce]. The prepared iron oxide/ceria nanoparticle structure solution was transferred to microplate wells three times (200 μL each). After adding xanthine oxidase (20 μL) to each well, the microplate was incubated at 37° C. for 20 minutes. SOD-like activity was measured by measuring the absorbance at 450 nm of each well, and 50 U/mL SOD was defined as the amount of SOD that inhibited the reduction reaction of WST-1 by 50%.

CAT-like activity was measured using a CAT assay kit (Amplex Red hydrogen peroxide/peroxidase assay kit, Molecular Probes, A22188). Iron oxide/ceria nanoparticle structures were diluted to different concentrations (0, 0.375, 0.75 and 1.5 mM [Ce]) in reaction buffer containing 100 μM Amplex Red reagent and 2 mM hydrogen peroxide. 50 μL of each solution was transferred three times to the microplate wells. After incubation at room temperature for 30 minutes, the absorbance of each well at 490 nm was measured. 1 mU/mL horseradish peroxidase (HRP) was used as a 100% control when measuring CAT-like activity.

cell culture

HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS). ^{Cells were maintained at 1×10 5} cells/mL at 37° C. in a humidified atmosphere of 5% CO _{2 .}

Cell viability assay

HeLa cells were seeded in 96-well plates (10,000 cells/well) and incubated for 12 hours. 100 μL each of iron oxide/ceria nanoparticle constructs diluted in cell culture medium were added to the microplate wells to give final concentrations of 0, 0.06, 0.125, 0.25, 0.5 and 1 mM [Fe] (0, 2.38, 4.75, 9.5, respectively) 19 and 38 μM [Ce]). Cells were incubated at 37° C. for 24 hours, and 20 μL of MTT (3-[4,5-dimethylthialzol-2-yl]-2,5-diphenyltetrazolium bromide) (5 mg/mL) was added to each well. After incubating the cells at 37° C. for 4 hours, dimethyl sulfoxide (200 μL) was added to each well. Cell viability was determined by measuring the absorbance of each well at 595 nm.

Blood Amyloid-β Peptide Cleansing

2 month old 5XFAD transgenic male mice were anesthetized with isofluorane. A 29 gauge needle connected to a medical tube (inner diameter = 0.5 mm) was inserted into the femoral vein of the mouse and used as a blood drain point. The inner side of the needle and tube were pretreated with 2% heparin in PBS for 8 hours to prevent blood clotting during the cleansing process.

Blood circulation through the tube was initiated by a peristaltic pump at a flow rate of 150 μL/min. Simultaneously, a PBS solution of iron oxide/ceria nanoparticulate constructs (1.8 mM [Fe]) was introduced into the blood by a syringe pump at a flow rate of 10 μL/min through a three-way micromixer connected close to the blood outlet point. The other end of the tube was connected to a 31 gauge needle inserted into the jugular vein of the mouse, and the treated blood was injected back into the body. A neodymium magnet is positioned near the end of the extracorporeal blood circuit, a blood injection point, to separate the iron oxide/ceria nanoparticle structure and the amyloid-β peptide bound thereto. Blood cleansing was performed for 0.5 min per gram of mouse body weight.

Plasma Amyloid-β Immunoassay

Quantification was performed using a human amyloid-β enzyme-linked immunosorbent assay (ELISA) kit (R & D systems, DAB142). Blood samples from each mouse were collected into anticoagulant-treated microtubes before and after blood cleansing treatment (approximately 50 μL each). After cell removal by centrifugation, the resulting supernatant was diluted 10-fold with diluent buffer (RD2-7) and analyzed. Measurements were performed in triplicate.

western blot

Plasma samples obtained before and after blood cleansing treatment were diluted 1,000-fold with PBS. Samples were denatured at 97° C. for 5 minutes and cooled on ice for 10 minutes. After centrifugation, 10 μl of each supernatant was loaded onto an SDS-PAGE gel. The gel was transferred onto a nitrocellulose membrane. The membrane was blocked after washing with 5% skim milk in 0.1M PBS containing 0.05% Tween 20 (PBST) for 1 hour.

Anti-amyloid-β antibody (BioLegend, 800702) diluted with PBST containing 5% skim milk (1:1000) was reacted overnight. After washing with 5% skim milk in PBST, an HRP-conjugated goat polyclonal anti-mouse antibody (Abcam, ab6789) was used as a secondary antibody. After rinsing, HRP substrate reagent (Merck Millipore, WBLUR0500) was applied at room temperature for 2 minutes. Chemiluminescence signals were captured for analysis.

Plasma reactive oxygen species analysis

ROS/RNS assay kit (Cell Biolabs, STA-347) was used to compare reactive oxygen species levels. Plasma samples obtained before and after the blood cleansing treatment were diluted 100-fold with PBS, and 50 μL of each was transferred to a 96-well microplate. 50 μl of the diluted catalyst (Part No. 234703) solution was added to each well. After incubation at room temperature for 5 minutes, 100 μL of a diluted dichlorodihydrofluorescein (Part No. 234704) solution was added to each well and incubated for 15 minutes. Fluorescence at 530 nm was measured using 480 nm excitation. Measurements were performed in triplicate.

Brain Amyloid-β Immunoassay

2 month old 5XFAD transgenic male mice received blood amyloid-β peptide cleansing treatment. Mice were grown in the same sterile laboratory conditions for one month before receiving a second blood amyloid-β peptide cleansing treatment on another part of the body.

After one more month of growth, mice _{were euthanized with CO 2} and blood was collected from myocardial infarction in anticoagulant-treated CBC tubes for blood analysis. Mice were perfused with 4% paraformaldehyde in PBS and decapitated for brain removal. The left cerebral hemisphere of each brain was dissolved in 1.5 mL of a radioimmunoprecipitation assay (RIPA) buffer containing 1% protease inhibitor cocktail (Cell Biolabs, AKR-190). After centrifugation, the supernatant was divided and stored at -80°C until use.

For amyloid-β quantification, brain lysates were diluted 5-fold with dilution buffer (RD2-7) and analyzed using a human amyloid-β ELISA kit (R & D systems, DAB142). The amyloid-β concentration of each brain lysate measured was normalized to the concentration of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the same brain lysate. For GAPDH measurement, the brain lysate was diluted 10,000-fold and analyzed using a GAPDH ELISA kit (R&D systems, DYC5718). All measurements were performed in triplicate.

Immunohistofluorescence

The right cerebral hemisphere of the acquired brain was fixed in 0.1 M phosphate buffer containing 4% paraformaldehyde at 4 °C for 20 h and then cut into 30 µm sections using a cryostat for 72 h at 4 °C with 30% sucrose. was stored in 0.05 M PBS containing The prepared tissue sections were placed on a glass slide, immersed in acetone at -20°C for 10 minutes, and washed with PBST. Tissue sections were blocked with blocking buffer (ThermoFisher, 37538) containing 0.05% Tween-20 at room temperature for 1 hour.

Tissue sections were rinsed with PBST and incubated with anti-amyloid-β antibody (1:1000, BioLegend, 800702) or anti-GFAP antibody (1:200, ThermoFisher MA5-12023) for 2 h. After washing with PBST, Alexa Fluor 594-conjugated polyclonal anti-mouse antibody (1:200, ThermoFisher R37121) was added as a secondary antibody and incubated for 1 hour. Tissue sections were washed again and cortical fluorescence was observed under CLSM.

So far, specific embodiments of the present invention have been described. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

1: Blood Cleansing System
10: blood circulation unit 11: blood circulation pump
12: blood discharge tube 13: blood inlet tube
20: nanoparticulate structure supply unit 30: nanoparticulate structure recovery unit
100: nanoparticle structure 110: core
111: iron oxide nanoparticles 120: shell
121: ceria nanoparticles 130: dispersible compound
140: antibody

Claims (9)

a core comprising a cluster to which magnetic nanoparticles are assembled; and
It is located on the surface of the cluster and comprises a shell including ceria nanoparticles,
The cluster is formed by self-assembly of the magnetic nanoparticles, and any one of the magnetic nanoparticles is in contact with at least one other magnetic nanoparticles among the magnetic nanoparticles,
The shell is a nanoparticle structure, characterized in that formed by coating the ceria nanoparticles on the cluster. The method of claim 1,
The magnetic nanoparticles are nanoparticle structures, characterized in that it comprises iron oxide nanoparticles. delete delete delete The method of claim 1,
Further comprising a dispersible compound bound to the ceria nanoparticles,
The dispersible compound is a nanoparticulate structure, characterized in that providing dispersibility to the nanoparticle structure. 7. The method of claim 6,
The dispersible compound is a nanoparticulate structure, characterized in that it comprises PEG. The method of claim 1,
The nanoparticle structure is a nanoparticle structure, characterized in that it has a hydrodynamic diameter of 200 ~ 400nm. The method of claim 1,
The shell is a nanoparticle structure, characterized in that the single layer of the ceria nanoparticles.

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