MAGNETIC IMMUNO-PARTICLE AND USE THEREOF

Abstract

Provided are magnetic immunoparticles and use thereof, specifically, magnetic immunoparticles including a cell membrane capable of capturing a pathogenic material and magnetic particles attached to the cell membrane, a method of detecting pathogenic materials using the magnetic immunoparticles, and a method of diagnosing and treating an infectious disease using the magnetic immunoparticles. The magnetic immunoparticles according to an aspect may include cell membranes capable of capturing pathogenic materials, and thus may minimize side effects in vivo, and may detect various kinds of pathogenic materials due to characteristics of the cells from which the cell membranes are derived. Further, since the magnetic immunoparticles include magnetic particles, the magnetic immunoparticles may be easily separated by applying a magnetic field, and thus pathogenic materials may be more effectively detected and removed.

Claims

1. Magnetic immunoparticles comprising: a cell membrane capable of capturing a pathogenic material; and magnetic particles attached to the cell membrane, wherein the cell membrane is derived from one or more selected from the group consisting of immune cells, red blood cells, endothelial cells, and epithelial cells.

2. The magnetic immunoparticles of claim 1, wherein the pathogenic material is one or more selected from the group consisting of pathogenic bacteria, fungi, viruses, parasites, prions, and toxins.

3. The magnetic immunoparticles of claim 1, wherein the immune cells are one or more selected from the group consisting of neutrophils, eosinophils, basophils, monocytes, lymphocytes, Kupffer cells, microglias, macrophages, dendritic cells, mast cells, B cells, T cells, natural killer cells (NK cells), immune cell-derived cell lines, immune cell-like cells, and stem cell-derived immune cells.

4. The magnetic immunoparticles of claim 1, wherein the magnetic particles comprise one or more magnetic elements selected from the group consisting of iron (Fe), nickel (Ni), cobalt (Co), manganese (Mn), bismuth (Bi), zinc (Zn), strontium (Sr), lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), ruthenium (Lu), copper (Cu), silver (Ag), gold (Au), cadmium (Cd), mercury (Hg), aluminum (Al), gallium (Ga), indium (in), thallium (TI), calcium (Ca), barium (Ba), radium (Ra), platinum (Pt), and lead (Pd).

5. The magnetic immunoparticles of claim 4, wherein the magnetic elements are oxidized or surface-modified with metals, functional groups, proteins, carbohydrates, polymers, or lipids.

6. The magnetic immunoparticles of claim 1, wherein the magnetic particles are comprised in a solution.

7. The magnetic immunoparticles of claim 1, comprising an outer surface comprising the cell membrane and an inner core comprising the magnetic particles.

8. The magnetic immunoparticles of claim 7, wherein the inner core comprises one or more magnetic particles.

9. The magnetic immunoparticles of claim 1, wherein the cell membrane forms a vesicle.

10. The magnetic immunoparticles of claim 1, wherein the cell membrane expresses one or more selected from the group consisting of lectins, Toll like receptors (TLRs), pattern recognition receptors (PRRs), cluster of differentiation (CD) molecules, neutrophil extracellular traps (NETs), glycophorins, and cytokine receptors.

11. The magnetic immunoparticles of claim 1, wherein the magnetic immunoparticles are used to detect or remove pathogenic materials.

12. A method of diagnosing an infectious disease, the method comprising bringing the magnetic immunoparticles of claim 1 into contact with a sample and mixing the magnetic immunoparticles with the sample, and applying a magnetic field to the mixed sample.

13. The method of claim 12, further comprising detecting pathogenic materials bound to the magnetic immunoparticles, wherein the pathogenic materials are one or more selected from the group consisting of pathogenic bacteria, fungi, viruses, parasites, prions, and toxins.

14. The method of claim 12, wherein the infectious disease is one or more selected from the group consisting of systemic or local infections, inflammation, sepsis, and poisoning by toxins.

15. A method of treating an infectious disease, the method comprising bringing the magnetic immunoparticles of claim 1 into contact with a sample and mixing the magnetic immunoparticles with the sample, and removing a pathogenic material by applying a magnetic field to the mixed sample.

16. The method of claim 15, wherein the pathogenic material is one or more selected from the group consisting of pathogenic bacteria, fungi, viruses, parasites, prions, and toxins.

17. The method of claim 15, wherein the infectious disease is one or more selected from the group consisting of systemic or local infection, Inflammation, sepsis, and poisoning by toxins.

18. The method of claim 15, wherein hemodialysis or extracorporeal circulation is applied to the method of treating an infectious disease.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0120] The above and other aspects, features, and advantages of certain embodiments of the disclosure will be more apparent from the following description taken in conjunction with the accompanying drawings, in which:

[0121] FIG. 1 shows a schematic illustration of a method of producing magnetic immunoparticles according to one exemplary embodiment;

[0122] FIG. 2 shows transmission electron microscope (TEM) images of magnetic immunoparticles produced according to one exemplary embodiment, wherein the left image shows magnetic particles used in one exemplary embodiment, and the right image shows magnetic immunoparticles produced according to one exemplary embodiment;

[0123] FIG. 3 is a graph showing a bacteria removal rate (%) by using the magnetic immunoparticles according to one exemplary embodiment, wherein D-HL represents the use of cell membranes of differentiated HL60 cells, and C-HL represents the use of cell membranes of undifferentiated HL60 cells, E+V as a comparative group represents an experimental group treated with cell membranes to which magnetic particles had not been attached, and E+V+M represents an experimental group treated with magnetic immunoparticles to which magnetic particles had been attached;

[0124] FIG. 4 shows a schematic illustration of a method of collecting, concentrating, and removing pathogenic materials in blood, based on the magnetic immunoparticles according to one exemplary embodiment;

[0125] FIG. 5 is a graph showing a removal rate (%) of MRSA, which is Gram-positive bacteria, by using the magnetic immunoparticles according to one exemplary embodiment;

[0126] FIG. 6 is a graph showing a removal rate (%) of ESBL-EC, which is Gram-negative bacteria, by using the magnetic immunoparticles according to one exemplary embodiment;

[0127] FIG. 7 is a graph showing a removal rate (%) of HCoV229E virus by using the magnetic immunoparticles according to one exemplary embodiment;

[0128] FIG. 8 is a graph showing the ability of the magnetic immunoparticles according to one exemplary embodiment to remove pathogenic bacteria (MRSA) in diabetic blood;

[0129] FIG. 9 is a graph showing the ability of the magnetic immunoparticles according to one exemplary embodiment to remove a pathogenic virus (CMV) in diabetic blood;

[0130] FIG. 10 shows a schematic illustration of a method of removing pathogenic materials by magnetic immunoparticle-based extracorporeal circulation according to one exemplary embodiment;

[0131] FIG. 11 is a graph showing the ability to remove pathogenic bacteria (MRSA) in blood in vitro at the time of using the method of removing pathogenic materials by magnetic immunoparticle-based extracorporeal circulation according to one exemplary embodiment;

[0132] FIG. 12 is a graph showing the ability to remove a pathogenic virus (CMV) in blood in vitro when using the method of removing pathogenic materials by magnetic immunoparticle-based extracorporeal circulation according to one exemplary embodiment;

[0133] FIG. 13 is a graph showing the ability to remove a pathogenic material (LPS) in blood in vitro when using the method of removing pathogenic materials by magnetic immunoparticle-based extracorporeal circulation according to one exemplary embodiment;

[0134] FIG. 14 is a graph showing the ability to remove a pathogenic material (ZIKV protein) in blood in vitro when using the method of removing pathogenic materials by magnetic immunoparticle-based extracorporeal circulation according to one exemplary embodiment;

[0135] FIG. 15 is a graph showing the ability to remove a pathogenic material (SARS-CoV-2 Spike protein) in blood in vitro when using the method of removing pathogenic materials by magnetic immunoparticle-based extracorporeal circulation according to one exemplary embodiment;

[0136] FIG. 16 shows a schematic illustration of the application, to an animal, of the method of removing pathogenic materials by magnetic immunoparticle-based extracorporeal circulation according to one exemplary embodiment;

[0137] FIG. 17 is a graph showing the ability to remove pathogenic bacteria (MRSA) in blood of a rat animal in vivo when using the method of removing pathogenic materials by magnetic immunoparticle-based extracorporeal circulation according to one exemplary embodiment;

[0138] FIG. 18 shows a schematic illustration of a magnetic immunoparticle-based extracorporeal circulation method using a fluidic device 100, in which P represents a pump;

[0139] FIG. 19 shows a schematic illustration of an example of the fluidic device 100;

[0140] FIG. 20 shows a schematic illustration of another example of the fluidic device 100A;

[0141] FIG. 21 shows a schematic illustration of a magnetic immunoparticle-based extracorporeal circulation machine 1000 for removing pathogenic materials, the machine including the fluidic device 100; and

[0142] FIG. 22 shows a schematic illustration of a magnetic immunoparticle-based extracorporeal circulation machine 1000′ for removing pathogenic materials, the machine including the fluidic device 100.

DETAILED DESCRIPTION

[0143] Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. In this regard, the present embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the embodiments are merely described below, by referring to the figures, to explain aspects of the present description. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. Expressions such as “at least one of,” when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.

[0144] Hereinafter, the present disclosure will be described in more detail with reference to exemplary embodiments. However, these exemplary embodiments are for illustrative purposes only, and the scope of the present disclosure is not intended to be limited by these exemplary embodiments. It is apparent to those skilled in the art that various changes may be made therein without departing from the spirit and scope of the present disclosure.

EXAMPLES

Example 1: Preparation of Magnetic Immunoparticles

[0145] Magnetic immunoparticles were prepared using human red blood cells in the blood of a human living body as a model. Red blood cells were obtained from the Red Cross (South Korea). Red blood cells were suspended in 1 mL of a 25% v/v mixture of PBS (pH 7.2, Biosesang, South Korea) and distilled water (Biosesang, South Korea) at a density of 10.sup.8 cells, and treated with a low osmotic pressure at 4° C. for 1 hour, and then centrifuged at 4° C. for 5 minutes (Centrifuge 5424R, Eppendorf, Germany) and prepared in 1×PBS.

[0146] In addition, cell membranes separated (purified) by the low osmotic pressure treatment were subjected to sonication (0700 Ultra-Sonicator, Qsonica, USA) for 10 minutes at 4° C. 20 kHz. and 150 W to split the cell membranes into smaller pieces.

[0147] Further, the prepared red blood cell-derived cell membranes were extruded together with magnetic particles in an Avanti mini extruder (Avanti Polar Lipids, Alabaster, Ala., USA) using 1 μm, 0.4 μm, and 0.2 μm pore size track-etched membrane filters sequentially to prepare magnetic immunoparticles. In detail, according to Type 2 as shown in FIG. 1, the prepared cell membranes and magnetic particles (0.5 mg/mL) were mixed and extruded to prepare magnetic immunoparticles in which the magnetic particles were invaginated into the cell membranes. As the magnetic particles, iron oxide magnetic particles (Carboxyl-Adembeads 200 nm, Ademtech, France) having an average particle size of 200 nm, of which surface was modified with carboxylic acid, were used.

[0148] As a result, as shown in FIG. 2, transmission electron microscope (TEM) images showed that magnetic immunoparticles (right image of FIG. 2) having an average size of about 250 nm were generated, in which the magnetic particles were invaginated into the cell-derived membranes.

Example 2: Test of Microbial Removal Ability of Magnetic Immunoparticles

[0149] E. coli (1×10.sup.8 CFU/mL) was inoculated in a solution containing the magnetic immunoparticles prepared in Example 1 and reacted for 2 hours at 15 rpm in an incubator at a temperature of 37° C.

[0150] Each reacted solution was transferred to a 1 mL tube (Eppendorf), and then a permanent magnet was attached to one side of the outer surfaces of the tube to apply a magnetic field to the tube for 40 minutes. After applying the magnetic field for 40 minutes, the solution was extracted from the tube on the opposite side of the permanent magnet attached to the tube, and each 100 μl of the extracted solution was plated on an LB agar plate, and the number of bacterial colonies was observed and quantified after overnight incubation. For comparison, magnetic immunoparticles prepared using undifferentiated HL60 cells or differentiated HL60 cells prepared in Example 1, differentiated HL60 cells or undifferentiated HL60 cells were used to allow the reaction of Example 2, respectively. A reduction rate (%) was expressed as a percentage of (1-(the number of E. coli after treatment with magnetic immunoparticles/the number of E. coli before treatment with magnetic immunoparticles)).

[0151] As a result, as shown in FIG. 3, it was confirmed that when the magnetic immunoparticles prepared by using the cell membrane of differentiated HL60 cells were used, the E. coli removal rate was about 75%, indicating an excellent microbial removal efficiency.

Example 3-Example 12: Preparation of Magnetic Immunoparticles Using Various Cells

[0152] In the present exemplary embodiments, various kinds of magnetic immunoparticles were prepared using the following cells according to the method of Example 1:

[0153] human red blood cell (RBC); human U937-differentiated M0 macrophage: M0 macrophage-like cell obtained by differentiating human U937 cell line (leukemia cell line): human U937-differentiated M1 macrophage: M1 macrophage-like cell obtained by differentiating human U937 cell line; human U937-differentiated M2 macrophage: M2 macrophage-like cell obtained by differentiating human U937 cell line; human THP-1-differentiated M0 macrophage: M0 macrophage-like cell obtained by differentiating human THP-1 cell line (human monocyte cell line derived from a patient with acute monocytic leukemia); human HL-60-differentiated neutrophil: neutrophil-like cell obtained by differentiating human HL-60 cell line (leukemia cell line); human K562 cell line (leukemia cell line); human oral epithelial cell; human hepatic sinusoidal endothelial cell (HSEC); or human intestinal epithelial cell line (Caco-2).

[0154] As a result, as shown in Table 1 below, a total of 10 types of magnetic immunoparticles were obtained using cell membranes derived from the above various cells.

TABLE-US-00001 TABLE 1 Magnetic immunoparticles Cell membrane Example 3 Cell membrane of human RBC Example 4 Cell membrane of human U937-differentiated M0 macrophage Example 5 Cell membrane of human U937-differentiated M1 macrophage Example 6 Cell membrane of human U937-differentiated M2 macrophage Example 7 Cell membrane of human THP-1-differentiated M0 macrophage Example 8 Cell membrane of human HL-60-differentiated neutrophil Example 9 Cell membrane of human K562 Example 10 Cell membrane of human hepatic sinusoidal endothelial cell (HSEC) Example 11 Cell membrane of human intestinal epithelial cell (Caco-2) Example 12 Cell membrane of human oral epithelial cell

EXPERIMENTAL EXAMPLES

Experimental Example 1: Test of Removal Ability of Magnetic Immunoparticles Against Gram-Positive/Negative Bacteria

[0155] In this Experimental Example, to examine whether the magnetic immunoparticles of Table 1 are able to remove pathogens in the blood, each of the magnetic immunoparticles of Table 1 was independently injected into a human blood sample containing bacteria and the bacteria captured by the magnetic immunoparticles were removed by applying a magnetic field, and then changes of colony forming unit (CFU) of the inoculated bacteria in the sample were measured.

[0156] In detail, methicillin resistant Staphylococcus aureus (MRSA) which is a Gram-positive bacterium or extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-EC) which is a Gram-negative bacterium was inoculated in 1 mL of an anticoagulant-treated human blood (Red Cross, South Korea) sample at a concentration of 10.sup.4 CFU/mL, and incubated at 37° C. for 10 minutes. Each of the magnetic immunoparticles of Table 1 was independently injected into the incubated blood sample such that the final concentration of the magnetic immunoparticles was 150 μg/mL. The equivalent amount of physiological saline was injected into a control group. Thereafter, after a reaction for 20 minutes at 37° C., the magnetic immunoparticles in the blood sample were fixed at a specific position using a magnet for 15 minutes to prevent the magnetic immunoparticles from being included in the supernatant, and then the supernatant was collected. CFU of bacteria in the supernatant was examined. In detail, the supernatant (100 μL) of the blood sample was diluted with 900 μL of physiological saline, and plated on LB agar medium using a microbial analyzer (EDDY JET2, IUL micro, USA), and incubated at 37° C. for 24 hours. Thereafter, CFU of the bacteria on the LB agar medium was measured using a microbial colony counter (Sphereflash colony counter and zone reader, IUL micro, USA).

[0157] As a result, as shown in FIGS. 5 and 6, it was confirmed that most of the magnetic immunoparticles are able to remove MRSA and ESBL-EC in the blood sample by capturing. In particular, it was confirmed that magnetic immunoparticles (Example 3) prepared using cell membranes of human red blood cells (RBC) showed the most excellent removal ability against MRSA and ESBL-EC in blood samples. In detail, it was confirmed that the magnetic immunoparticles (Example 3) prepared using the cell membrane of human red blood cells (RBC) are able to remove about 75% or more of MRSA Gram-positive bacterium or about 85% or more of Gram-negative bacteria ESBL-EC in the blood sample.

Experimental Example 2: Test of Removal Ability of Magnetic Immunoparticles Against Viruses

[0158] In this Experimental Example, to examine whether the magnetic immunoparticles of Table 1 are able to remove viruses in the blood, each of the magnetic immunoparticles of Table 1 was independently injected into a human blood sample containing viruses, and the viruses captured by the magnetic immunoparticles were removed by applying a magnetic field, and then changes in the amounts of RNA of the inoculated viruses in the culture medium were measured.

[0159] In detail, HCoV229E (Human Coronavirus 229E) was inoculated in 1 mL of an anticoagulant-treated human blood (Red Cross, South Korea) sample at a density of 10′ PFU/mL, and incubated at 37° C. for 10 minutes. Each of the magnetic immunoparticles of Table 1 was independently injected into the incubated blood sample such that the final concentration of the magnetic immunoparticles was 150 μg/mL. The equivalent amount of physiological saline was injected into a control group. Thereafter, after a reaction for 20 minutes at 37° C., the magnetic immunoparticles in the blood sample were fixed at a specific position using a magnet for 15 minutes to prevent the magnetic immunoparticles from being included in the supernatant, and then the supernatant was collected. The amount of RNA of viruses in the supernatant was examined. Nucleic acids were extracted from viruses in the supernatant using a QIAmp viral RNA mini kit (QIAGEN, Germany), and the extracted nucleic acids were amplified using SYBR PCR master mix (Toyobo, Japan) and Real time PCR (CFX connect, BIO-RAD, USA) to measure the amount of RNA.

[0160] As a result, as shown in FIG. 7, it was confirmed that the magnetic immunoparticles (Example 3) prepared using the cell membrane of human red blood cells (RBC), the magnetic immunoparticles (Example 10) prepared using the cell membrane of human hepatic sinusoidal endothelial cells (HSEC), and the magnetic immunoparticles (Example 8) prepared using the cell membrane of human HL-60-differentiated neutrophil are able to remove HCoV229E virus in the blood sample by capturing. In particular, it was confirmed that magnetic immunoparticles (Example 10) prepared using cell membranes of human hepatic sinusoidal endothelial cells (HSEC) showed the most excellent removal ability against HCoV229E virus in the blood sample. In detail, it was confirmed that magnetic immunoparticles (Example 10) prepared using the cell membrane of human hepatic sinusoidal endothelial cells (HSEC) are able to remove about 70% or more of HCoV229E virus in the blood sample by capturing.

Experimental Example 3: Test of Removal Ability of Magnetic Immunoparticles Against Pathogens (Bacteria or Viruses) in Diabetic Blood

[0161] In this Experimental Example, to examine whether the magnetic immunoparticles of Table 1 are able to remove pathogens (bacteria or viruses) in the diabetic blood, pathogens were arbitrarily inoculated into a human blood sample to which glucose (D-glucose, Sigma-Aldrich, USA) was arbitrarily added, and then cultured. Each of the magnetic immunoparticles of Table 1 were independently injected to the culture medium, and the pathogens captured by the magnetic immunoparticles were removed by applying a magnetic field. Thereafter, changes in the concentration of the pathogens in the culture medium were measured.

[0162] In detail, D-glucose was added in 1 mL of an anticoagulant-treated human blood (Red Cross, South Korea) sample at a concentration of about 400 mg/dL to about 450 mg/dL, and incubated at 37° C. for 10 minutes. Pathogens (bacteria or viruses) were inoculated in the incubated blood sample at a concentration of 10.sup.4 CFU/mL (or 105 PFU/mL), and incubated at 37° C. for 10 minutes. Each of the magnetic immunoparticles of Table 1 was independently injected into the incubated blood sample such that the final concentration of the magnetic immunoparticles was 150 μg/mL. The equivalent amount of physiological saline was injected into a control group. Thereafter, after a reaction for 20 minutes at 37° C., the magnetic immunoparticles in the blood sample were fixed at a specific position using a magnet for 15 minutes to prevent the magnetic immunoparticles from being included in the supernatant, and then the supernatant was collected. The concentration of the pathogens in the supernatant was examined. Changes in the concentration of bacteria, among the pathogens, in the blood sample were determined by measuring CFU of the bacteria in the same manner as in Experimental Example 1, and changes in the concentration of viruses, among the pathogens, in the blood sample were determined by measuring the amount of RNA of the viruses in the same manner as in Experimental Example 2. As the pathogens inoculated in this Experimental Example, MRSA or Cytomegalovirus (CMV) was used, and as the injected magnetic immunoparticles, the magnetic immunoparticles (Example 3) prepared using the cell membrane of human red blood cell (RBC), the magnetic immunoparticles (Example 12) prepared using the cell membrane of human oral epithelial cell, and the magnetic immunoparticles (Example 10) prepared using the cell membrane of human hepatic sinusoidal endothelial cell (HSEC) were used.

[0163] As a result, as shown in FIG. 8, it was confirmed that the concentration of the MRSA pathogen in the diabetic blood sample was remarkably reduced by injecting the magnetic immunoparticles (Example 3) prepared using the cell membrane of human red blood cell (RBC), as compared with the control group, and the concentration of the MRSA pathogen was further reduced as the removal process was repeated.

[0164] As shown in FIG. 9, it was also confirmed that the concentration of the CMV pathogen in the diabetic blood sample was remarkably reduced by injecting the magnetic immunoparticles (Example 12) prepared using the cell membrane of human oral epithelial cell and the magnetic immunoparticles (Example 10) prepared using the cell membrane of human hepatic sinusoidal endothelial cell (HSEC), and the concentration of the CMV pathogen was further reduced as the removal process was repeated.

[0165] This Experimental Example confirmed that the magnetic immunoparticles of Table 1 are able to effectively remove pathogens (bacteria or viruses) in the diabetic blood.

Experimental Example 4: In Vitro Removal of Pathogens or Pathogenic Materials in Blood by Method of Removing Pathogenic Materials Using Magnetic Immunoparticle-Based Extracorporeal Circulation

[0166] In this Experimental Example, for in vitro removal of pathogens or pathogenic materials in a large amount of blood using the magnetic immunoparticles of Table 1, a method of removing pathogenic material using magnetic immunoparticle-based extracorporeal circulation was used.

[0167] The method of removing pathogenic material using magnetic immunoparticle-based extracorporeal circulation includes, as shown in FIG. 10, binding the pathogens or pathogenic materials with the magnetic immunoparticles during a process of mixing the pathogens or pathogenic material-contaminated blood with the magnetic immunoparticles in a reaction unit (a fluid element for mixing) of the magnetic immunoparticle-based extracorporeal circulation machine for removing pathogenic materials; and removing the complex bound to the pathogens or pathogenic materials to the magnetic immunoparticles from the blood by a magnet during a process of passing the blood including the complex through a magnetic field-forming unit (a fluid element for magnetophoretic separation). Blood may be purified by repeating the process in which each procedure is sequentially performed in vitro.

[0168] In detail, bacteria (10.sup.4 CFU/mL), viruses (PFU/mL), or inflammatory materials (LPS, 10 μg/mL) were inoculated in 10 mL of anticoagulant-treated human blood (Red Cross, South Korea) or whole blood of a rat (8-week-old, male), and incubated at 37° C. for 10 minutes. In addition, a solution containing the magnetic immunoparticles of Table 1 was prepared in saline at a concentration of 0.5 mg/mL. The incubated blood sample and the prepared magnetic immunoparticle solution were injected into a magnetic immunoparticle-based extracorporeal circulation machine for removing pathogenic materials, and loaded at a rate of 10 mL/hr and 0.5 mL/hr, respectively. When the injected blood sample and the magnetic immunoparticle solution were mixed while running through the reaction unit (the fluid element for mixing), the pathogens or pathogenic materials in the blood sample were bound to the magnetic immunoparticles. The complexes bound to the pathogens or pathogenic materials to the magnetic immunoparticles in the blood were captured toward the magnet by a magnetic field while passing through the magnetic field-forming unit (fluid element for magnetophoretic separation), such that the complexes were removed from the blood sample. The blood sample from which the pathogens or pathogenic materials had been removed was collected, and then injected again into the magnetic immunoparticle-based extracorporeal circulation machine for removing pathogenic materials, and the process of removing pathogenic materials by the above magnetic immunoparticle-based extracorporeal circulation was repeated for 5 hours. At the same time, the concentrations of the pathogens or pathogenic materials in the blood sample were measured every hour. The change in the concentrations of bacteria, among pathogens, in the blood sample was determined by measuring CFU of the bacteria in the same manner as in Experimental Example 1, and the change in the concentrations of viruses, among pathogens, in the blood sample was determined by measuring the amount of RNA of the viruses in the same manner as in Experimental Example 2. In addition, changes in the concentrations of LPS, ZIKV Protein, or SARS-CoV-2 Spike Protein as pathogenic materials in the blood samples were determined by enzyme-linked immunosorbent assay (ELISA), and an LPS ELISA kit (LS-F55757-1, LSbio, USA), a Zika virus (strain Zika SPH2015) Envelope Protein (ZIKV-E) ELISA Kit (Sinobio, China), or a SARS-CoV-2 Spike protein ELISA kit (ab274342, abcam, USA) was used.

[0169] In this Experimental Example, when MRSA as a bacterial pathogen was inoculated in human blood or rat blood samples, in order to remove the MRSA, the magnetic immunoparticles (Example 3) prepared using the cell membrane of human red blood cells (RBC) or the magnetic immunoparticles prepared using the cell membrane of red blood cells of a Wistar rat (8 weeks old, male, Orient Bio, South Korea) were used as the magnetic immunoparticles.

[0170] In this Experimental Example, when CMV as a viral pathogen was inoculated in the blood sample, in order to remove the CMV, the magnetic immunoparticles (Example 10) prepared using the cell membrane of human hepatic sinusoidal endothelial cells (HSEC) were used as magnetic immunoparticles.

[0171] In this Experimental Example, when LPS as a pathogenic material was inoculated in the blood sample, in order to remove the LPS, the magnetic immunoparticles (Example 3) prepared using the cell membrane of human red blood cells (RBC) were used as magnetic immunoparticles.

[0172] In this Experimental Example, when ZIKV protein as a pathogenic material was inoculated in the blood sample, in order to remove the ZIKV protein, the magnetic immunoparticles (Example 10) prepared using the cell membrane of human hepatic sinusoidal endothelial cells (HSEC) or the magnetic immunoparticles (Example 9) prepared using the cell membrane of human K562 were used as magnetic immunoparticles.

[0173] In this Experimental Example, when SARS-CoV-2 Spike protein as a pathogenic material was inoculated in the blood sample, in order to remove the SARS-CoV-2 Spike protein, the magnetic immunoparticles (Example 10) prepared using the cell membrane of human hepatic sinusoidal endothelial cells (HSEC), the magnetic immunoparticles (Example 11) prepared using the cell membrane of human intestinal epithelial cells (Caco-2), or the magnetic immunoparticles (Example 7) prepared using the cell membrane of human THP-1-differentiated M0 macrophage were used as magnetic immunoparticles.

[0174] As a result, as shown in FIG. 11, it was confirmed that the magnetic immunoparticles (Example 3) prepared using the cell membrane of human red blood cells (RBC) or the magnetic immunoparticles prepared using the cell membrane of red blood cells of a rat are able to remove about 90% or more of MRSA in the human blood or rat blood.

[0175] As shown in FIG. 12, it was also confirmed that the magnetic immunoparticles (Example 10) prepared using the cell membrane of human hepatic sinusoidal endothelial cells (HSEC) are able to remove about 66% or more of CMV in the blood.

[0176] As shown in FIG. 13, it was also confirmed that the magnetic immunoparticles (Example 3) prepared using the cell membrane of human red blood cells (RBC) are able to remove about 90% or more of LPS in the blood.

[0177] As shown in FIG. 14, it was also confirmed that the magnetic immunoparticles (Example 10) prepared using the cell membrane of human hepatic sinusoidal endothelial cells (HSEC) or the magnetic immunoparticles (Example 9) prepared using the cell membrane of human K562 are able to remove about 50% or more of ZIKV protein in the blood.

[0178] As shown in FIG. 15, it was also confirmed that the magnetic immunoparticles (Example 10) prepared using the cell membrane of human hepatic sinusoidal endothelial cells (HSEC) are able to remove about 20% or more of SARS-CoV-2 Spike protein in the blood, the magnetic immunoparticles (Example 11) prepared using the cell membrane of human intestinal epithelial cells (Caco-2) are able to remove about 30% or more of SARS-CoV-2 Spike protein in the blood, and the magnetic immunoparticles (Example 7) prepared using the cell membrane of human THP-1-differentiated M0 macrophage are able to remove about 50% or more of SARS-CoV-2 Spike protein in the blood.

[0179] This Experimental Example confirmed that pathogens (bacteria or viruses) or pathogenic materials in a large amount of blood may be effectively removed in vitro using the method of removing pathogenic materials by extracorporeal circulation based on the magnetic immunoparticles of Table 1.

Experimental Example 5: In Vivo Removal of Pathogens or Pathogenic Materials in Blood by Method of Removing Pathogenic Materials Using Magnetic Immunoparticle-Based Extracorporeal Circulation

[0180] In this Experimental Example, for in vivo removal of pathogens or pathogenic materials in the blood using the magnetic immunoparticles of Table 1, a method of removing pathogenic material using magnetic immunoparticle-based extracorporeal circulation was used.

[0181] The method of removing pathogenic materials using magnetic immunoparticle-based extracorporeal circulation is the same as in Experimental Example 4, but is different from Experimental Example 4 in that the magnetic immunoparticle-based extracorporeal circulation machine for removing pathogenic materials was directly applied to a rat animal model arbitrarily infected with bacteria, and an in vivo test was performed in this Experimental Example.

[0182] In detail, as shown in FIG. 18, a catheter was inserted into the jugular vein of a normal Wistar rat (10-week-old, male) through surgery, and MRSA was arbitrarily injected through the catheter to infect the rat animal. In addition, a magnetic immunoparticle solution containing the magnetic immunoparticles prepared using the cell membrane of red blood cells derived from a Wistar rat (8-week-old, male) at a concentration of 0.5 mg/mL was prepared in saline. The infected rat and the magnetic immunoparticle-based extracorporeal circulation machine for removing pathogenic materials were connected through the catheter. By injecting the prepared magnetic immunoparticle solution into the extracorporeal circulation machine for removing pathogenic materials, which was connected to the rat, the method of removing pathogenic materials using magnetic immunoparticle-based extracorporeal circulation was performed, and the blood from which the pathogenic materials had been removed was injected back into the rat connected with the extracorporeal circulation machine for removing pathogenic materials.

[0183] The whole blood was collected from the rat at regular intervals (0 minutes, 15 minutes, 30 minutes, and 60 minutes) to measure changes in the concentrations of MRSA in the blood. The changes in the concentrations of MRSA was determined by measuring CFU of the bacteria in the whole blood sample collected in the same manner as in Experimental Example 1.

[0184] As a result, as shown in FIG. 17, it was confirmed that MRSA in the whole blood of the rat may be removed by directly applying the method of removing pathogenic materials using the magnetic immunoparticle-based extracorporeal circulation to the rat infected with MRSA.

[0185] This Experimental Example confirmed that pathogens (bacteria or viruses) or pathogenic materials in the blood may be effectively removed in vivo using the method of removing pathogenic materials by extracorporeal circulation based on the magnetic immunoparticles of Table 1, and as a result, infectious diseases may be treated.

[0186] Magnetic immunoparticles according to an aspect may include cell membranes derived from cells, and thus may minimize side effects in vivo, and may detect various kinds of pathogenic materials due to characteristics of the cells from which the cell membranes are derived. Further, since the magnetic immunoparticles include magnetic particles, the magnetic immunoparticles may be easily separated by applying a magnetic field, and thus pathogenic materials may be more effectively detected and removed. Furthermore, when the magnetic immunoparticles are used for treatment, the possibility of injection of the magnetic immunoparticles Into the body may be minimized, and thus side effects in vivo may be remarkably reduced.

[0187] It should be understood that embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should typically be considered as available for other similar features or aspects in other embodiments. While one or more embodiments have been described with reference to the figures, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the disclosure as defined by the following claims.