Extracorporeal blood purification device by removing reactive oxygen species and extracorporeal blood purification method using the same

Abstract

This specification discloses an extracorporeal blood purification device and an extracorporeal blood purification method using the same. In one aspect, the present invention uses porous microbeads loaded with ceria nanoparticles to effectively remove reactive oxygen species (ROS) in blood, which are substances that cause inflammation, outside the body. Specifically, the extracorporeal blood purification device is manufactured by loading the porous microbeads into a hemoperfusion cartridge and perfusing the patient's blood, thereby enabling rapid and widespread removal of reactive oxygen species in aqueous and blood conditions. Therefore, the present invention can be applied to a microbead-based extracorporeal hemoperfusion therapy system.

Description

[Technical Field]

[0001] This specification discloses an extracorporeal blood purification device and an extracorporeal blood purification method using the same.

[0002] This application was supported by the following research and development projects:

[0003] [Research and development project that supported this invention] [Assignment unique number]2022R1C1C100961012 [Department name] Ministry of Science, ICT and Technology of Korea [Management Agency] Korea Research Foundation [Project name] Basic Research Project in Science and Engineering > Support Project for New Research > Excellent New Research [Project title] Development of an extracorporeal blood purification system for sepsis treatment based on multifunctional microbeads [Contribution rate] 60 / 100 [Supervising institution] Seoul National University Bundang Hospital [Research period] March 1, 2022 to February 28, 2025

[0004] [Project unique number]RS-2023-00222910 [Department name] Ministry of Science, ICT and Technology of Korea [Management Agency] Korea Research Foundation [Project name] Bio and medical technology development (R&D) [Project title] Development of technologies applicable to medical practice for the five major diseases and training of scientists through a customized future medical research center in the era of 6P medicine [Contribution rate] 35 / 100 [Supervising institution] Seoul National University Bundang Hospital [Research period] April 1, 2023 - December 31, 2026

[0005] [Project unique number]13-2021-0004 [Department name] Seoul National University Bundang Hospital [Management Agency] Seoul National University Bundang Hospital [Project name] Joint research in key support areas [Project title] Development of extracorporeal blood purification device based on nanomaterials for cytokine storm treatment [Contribution rate] 5 / 100 [Supervising institution] Seoul National University Bundang Hospital [Research period] March 1, 2021 to March 1, 2024 [Background technology]

[0006] Sepsis is a severe infectious condition that essentially involves a systemic inflammatory response. Sepsis can progress to a life-threatening condition due to the body's excessive and misguided response to infection, resulting in tissue damage and organ dysfunction. Systemic inflammatory response syndrome (SIRS) is a systemic inflammatory response that the body exhibits in response to various forms of severe stress or injury, such as infection, trauma, burns, and pancreatitis. It plays an important pathophysiological role in sepsis. Various drugs have been developed and clinically tested to treat sepsis, but their efficacy has not been proven. Blood purification therapies (e.g., toraymyxin and cytosorb) that remove pathogen-related substances and cytokines extracorporeally have been developed as a new approach to sepsis treatment, but clinical trials have been unsuccessful, and their use is not recommended in clinical guidelines. Reactive oxygen species (ROS) are a crucial factor in SIRS, including sepsis, which, when excessively produced or inadequately removed, can damage cells and promote inflammation, leading to tissue damage and organ dysfunction. However, existing blood purification therapies have a clear limitation in that they cannot effectively remove reactive oxygen species. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide porous microbeads for extracorporeal hemoperfusion, an extracorporeal hemoperfusion cartridge containing the porous microbeads, or an extracorporeal blood purification apparatus containing the cartridge.

[0008] Another object of the present invention is to provide a method for producing the porous microbeads.

[0009] Another object of the present invention is to provide a method for extracorporeal blood purification using the porous microbeads. [Means for solving the problem]

[0010] To achieve the above object, in one aspect, the present invention provides porous microbeads for extracorporeal hemoperfusion containing ceria nanoparticles.

[0011] In one exemplary implementation, the porous microbeads can scavenge reactive oxygen species through the catalytic action of ceria nanoparticles.

[0012] In another aspect, the present invention provides an extracorporeal hemoperfusion cartridge comprising the porous microbeads.

[0013] In another aspect, the present invention provides an extracorporeal blood purification device comprising the above-described extracorporeal hemoperfusion cartridge.

[0014] In another aspect, the present invention provides a method for extracorporeal blood purification, comprising the steps of: (a) contacting blood isolated from a subject with the porous microbeads; (b) using the porous microbeads to remove reactive oxygen species from the blood; (c) recovering the blood from which the reactive oxygen species have been removed.

[0015] In another aspect, the present invention provides a method for producing porous microbeads for extracorporeal hemoperfusion. (a) synthesizing and surface modifying porous microbeads; (b) attaching ceria nanoparticles to the surface of the modified porous microbeads; [Effects of the Invention]

[0016] In one aspect, the present invention uses porous microbeads loaded with ceria nanoparticles to effectively remove reactive oxygen species (ROS) in blood, which are a cause of inflammation, outside the body. Specifically, an extracorporeal blood purification device is manufactured by loading the porous microbeads into a hemoperfusion cartridge and perfusing the patient's blood, thereby enabling rapid and widespread removal of reactive oxygen species in aqueous and blood conditions. Therefore, the present invention can be applied to a microbead-based extracorporeal hemoperfusion therapy system.

[0017] In another aspect, the present invention enables the continuous removal of reactive oxygen species. In particular, to solve the problem that continuous removal of reactive oxygen species is difficult under an extracorporeal perfusion environment, the present invention can achieve the continuous removal of reactive oxygen species by using a catalyst (ceria nanoparticles) instead of an adsorbent.

[0018] In another aspect, it is difficult to ensure the safety of inorganic nanoparticles such as ceria nanoparticles when injected into the body, making it difficult to develop them as drugs. However, the present invention has the advantage of ensuring safety by removing reactive oxygen species by perfusing blood outside the body.

[0019] In another aspect, the present invention can selectively remove reactive oxygen species, which is the fundamental problem in inflammatory and injury responses, and therefore can solve the problems of conventional extracorporeal blood perfusion devices, unlike conventional extracorporeal blood perfusion therapies, which nonspecifically adsorb all substances that may be useful to patients (e.g., cytokines).

[0020] In another aspect, the present invention can remove increased reactive oxygen species in severe diseases associated with systemic inflammatory response syndrome, including sepsis, by extracorporeal perfusion, thereby improving severity indicators (e.g., blood pressure, amount of vasopressors used, lactate levels) and increasing survival rates.

[0021] In another embodiment, the present invention can be applied to a microbead-based extracorporeal hemoperfusion therapy system that removes reactive oxygen species (ROS), which are substances that cause inflammation. [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 is a schematic diagram of a microbead-based hemoperfusion system for removal of reactive oxygen species in a sepsis model. [Figure 2] FIG. 1 is a diagram of a manufacturing process for porous silica microbeads. [Figure 3] 1 shows representative images of each step in the production of porous silica microbeads from porous silica particles / alginate microbeads. [Figure 4] Figure 1 shows nitrogen (N2) physisorption isotherms and pore size (squares: 1 wt%, triangles: 3 wt%, diamonds: 5 wt%) of the final porous silica microbeads as a function of the porous silica particle content (wt%) during the preparation of the initial porous silica particle / alginate microbeads. [Figure 5] FIG. 1 is a diagram of the steps for making ceria nanoparticles / porous silica microbeads by attaching ceria nanoparticles to the surface of porous silica microbeads by electrostatic attraction. [Figure 6] FIG. 1 is a diagram relating to the surface charge (zeta-potential) of porous silica particles and ceria nanoparticles. [Figure 7] 1 is a representative photograph of porous silica microbeads and ceria nanoparticles / porous silica microbeads with attached ceria nanoparticles. [Figure 8] FIG. 1 shows SEM images of porous silica microbeads and ceria nanoparticle / porous silica microbeads, and an analysis of the cerium (Ce) atomic distribution map on the surface of the microbeads by EDS. [Figure 9] Schematic diagram of ceria nanoparticles / porous silica microbeads with PVP-coated surfaces. [Figure 10] FIG. 1 shows the results of thermogravimetric analysis (TGA) of porous silica microbeads, ceria nanoparticle / porous silica microbeads, and PVP-coated ceria nanoparticle / porous silica microbeads. [Figure 11] Catalase-mimicking activity using porous silica microbeads, ceria nanoparticles / porous silica microbeads, and PVP-coated ceria nanoparticles / porous silica microbeads in a hydrogen peroxide / peroxidase assay incubated in H2O2 (500 μM). [Figure 12] FIG. 1 shows the hydroxyl radical scavenging activity of porous silica microbeads, ceria nanoparticles / porous silica microbeads, and PVP-coated ceria nanoparticles / porous silica microbeads using the HORAC assay kit. [Figure 13] FIG. 10 shows catalase mimetic activity as a function of the amount of ceria nanoparticles contained in the microbeads. [Figure 14] 1 is a representative image of a cartridge made for loading with microbeads. [Figure 15] 1 shows representative images of cartridges filled with porous silica microbeads, ceria nanoparticles / porous silica microbeads, and PVP-coated ceria nanoparticles / porous silica microbeads, respectively. [Figure 16] This figure shows the catalase-mimetic activity when hydrogen peroxide was passed through cartridges filled with 220 g of porous silica microbeads, ceria nanoparticles / porous silica microbeads, and PVP-coated ceria nanoparticles / porous silica microbeads, and the hydrogen peroxide that passed through the cartridge was collected and then repeatedly passed up to seven times. [Figure 17]FIG. 11 shows catalase mimetic activity measured by passing fresh hydrogen peroxide solution through a cartridge packed with PVP-coated ceria nanoparticles / porous silica microbeads and then collecting it. [Figure 18] This figure compares the catalase-mimetic properties of cartridges made by loading ceria nanoparticles onto non-pore glass microbeads and porous PES (polyethersulfone) polymer microbeads with those of a PVP-coated ceria nanoparticle / porous silica microbead cartridge. [Figure 19] 1 is a representative image of microbeads mixed with blood and then centrifuged to demonstrate the hemocompatibility of the microbeads. [Figure 20] FIG. 1 shows the degree of hemolysis based on absorbance analysis of the supernatant. [Figure 21] This is a representative image of microbeads that have been centrifuged, washed to remove blood, and then centrifuged again. [Figure 22] These are time-series images of blood being discharged from the body and perfused into the cartridge during an actual extracorporeal blood perfusion treatment. [Figure 23] FIG. 10 is a graph comparing the difference in hydrogen peroxide (H 2 O 2 ) levels released from leukocytes extracted from the blood between the hemoperfusion treatment group and the control group in sepsis-induced rats. [Figure 24] FIG. 10 is a graph comparing the difference in blood lactate levels, a marker of septic shock, between a group of rats with induced sepsis that underwent the hemoperfusion treatment and a control group. [Figure 25] FIG. 10 is a graph comparing the difference in survival rate between the hemoperfusion treatment group and the control group in sepsis-induced rats. [Figure 26] FIG. 10 is a graph comparing the difference in blood pressure between the hemoperfusion treatment group and the control group in sepsis-induced rats. [Figure 27]FIG. 10 is a graph comparing the difference in the amount of vasopressor used, which reflects the degree of shock, between the hemoperfusion treatment group and the control group in sepsis-induced rats. DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention will be described in detail below.

[0024] In one aspect, the present invention relates to porous microbeads for extracorporeal hemoperfusion, which contain ceria nanoparticles.

[0025] In one exemplary implementation, the porous microbeads can scavenge reactive oxygen species through the catalytic action of ceria nanoparticles.

[0026] In one exemplary embodiment, the size (diameter) of the porous microbeads may be, but is not limited to, 1 μm or more, 10 μm or more, 100 μm or more, 150 μm or more, 200 μm or more, 250 μm or more, 300 μm or more, 350 μm or more, 400 μm or more, 450 μm or more, or 1500 μm or less, 1400 μm or less, 1300 μm or less, 1200 μm or less, 1100 μm or less, 1000 μm or less, 900 μm or less, 800 μm or less, 700 μm or less, 650 μm or less, 600 μm or less, or 550 μm or less. For example, the size (diameter) of the porous microbeads may be 1 to 1500 μm, 10 to 1000 μm, or 100 to 1000 μm. The size (diameter) of the porous microbeads can be adjusted taking into consideration the size of the cartridge and effective hemoperfusion. Further, as a non-limiting example, the size (diameter) of the porous microbeads may be larger than a blood cell, for example, greater than 10 μm.

[0027] In one exemplary implementation, the reactive oxygen species may be at least one selected from the group consisting of, but not limited to, superoxide anion, hydrogen peroxide, hydroxyl radical, and combinations thereof.

[0028] In one exemplary implementation, the pore volume of the porous microbeads is between 0.1 and 13.5 cm. 3 / g, preferably 0.1 to 5.0 cm 3 / g, more preferably 0.5 to 1.5 cm 3 / g, but is not limited thereto. Porous microbeads having the above pore volume can effectively support ceria nanoparticles and remove reactive oxygen species.

[0029] In one exemplary implementation, the surface area of ​​the porous microbeads is between 100 and 1000 m 2 / g, preferably 150 to 600m 2 / g, more preferably 200 to 400m 2 / g, the porous microbeads having the above surface area can effectively support ceria nanoparticles and scavenge reactive oxygen species.

[0030] In one exemplary embodiment, the porous microbeads may contain ceria nanoparticles in an amount of 3 mg / g or more, specifically, 3 mg / g or more, 4 mg / g or more, 5 mg / g or more, 10 mg / g or more, 15 mg / g or more, 20 mg / g or more, 30 mg / g or more, 40 mg / g or more, or 50 mg / g or more, but not limited to, 500 mg / g or less, 400 mg / g or less, or 300 mg / g or less. For example, porous microbeads in a cartridge containing ceria nanoparticles in an amount of 100 mg / g or 200 mg / g can effectively perform extracorporeal hemoperfusion and removal of reactive oxygen species.

[0031] In one exemplary embodiment, the porous microbeads may be at least one selected from the group consisting of porous microbeads coated with alginate, porous microbeads coated with PEG (polyethylene glycol), porous microbeads coated with PVP (polyvinylpyrrolidone), and combinations thereof. The coating prevents the porous microbeads from reacting with blood and suppresses hemolysis or aggregation, thereby improving the blood compatibility of the porous microbeads. For this reason, porous microbeads coated with PEG (polyethylene glycol) or porous microbeads coated with PVP (polyvinylpyrrolidone) are preferred, and porous microbeads coated with PVP (polyvinylpyrrolidone) are more preferred.

[0032] In one exemplary implementation, the porous microbeads are made of mesoporous silica, styrene, C1-C4 alkyl-substituted styrene, vinylnaphthalene, vinylanthracene, cellulose, styrene-divinyl-benzene copolymer, polyethersulfone (PES), polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polyurethane (PUR), polyethylene terephthalate (PET), polystyrene (PS), ABS (Acrylonitrile butadiene styrene), polymethyl methacrylate (PMMA), polytetrafluoroethylene (PTFE), expanded polystyrene (Expanded Polystyrene), or the like. The microbeads may be, but are not limited to, microbeads comprising at least one selected from the group consisting of polystyrene (EPS), polycarbonate (PC), polyamide (PA), epoxy, unsaturated polyester (UP), melamine formaldehyde (MF), and combinations thereof. Suitable C1-C4 alkyl-substituted styrenes include, but are not limited to, ethylvinylbenzene, vinyltoluene, diethylstyrene, ethylmethylstyrene, or dimethylstyrene. It is understood that any of the various positional isomers of each of the above vinyl aromatic monomers is suitable.For example, the porous microbeads may be mesoporous silica microbeads, but any porous microbeads exhibiting the above-mentioned properties such as pore volume, surface area, and ability to support ceria nanoparticles may also be used, without limitation.

[0033] In another aspect, the present invention relates to an extracorporeal hemoperfusion cartridge comprising the porous microbeads.

[0034] In one exemplary embodiment, the volume fraction of the porous microbeads relative to the entire extracorporeal hemoperfusion cartridge may be 50% or more, 55% or more, 60% or more, 65% or more, or 70% or more, or 90% or less, 85% or less, or 80% or less. For example, the volume fraction of the porous microbeads relative to the entire extracorporeal hemoperfusion cartridge may be, but is not limited to, 50 to 90%. With this volume fraction, extracorporeal hemoperfusion and removal of active oxygen can be performed effectively.

[0035] In another aspect, the present invention relates to an extracorporeal blood purification device including the above-mentioned extracorporeal hemoperfusion cartridge.

[0036] In another aspect, the present invention relates to a method for extracorporeal blood purification, comprising the steps of: (a) contacting blood isolated from a subject with the porous microbeads; (b) using the porous microbeads to remove reactive oxygen species from the blood; (c) recovering the blood from which the reactive oxygen species have been removed.

[0037] In one exemplary embodiment, the step of removing reactive oxygen species may be performed for 2 to 24 hours, but is not limited thereto. The porous microbeads may use a catalyst (ceria nanoparticles) instead of an adsorbent, which allows for continuous removal of reactive oxygen. Therefore, reactive oxygen can be continuously removed for the required time without time constraints.

[0038] In another aspect, the present invention relates to a method for producing porous microbeads for extracorporeal hemoperfusion, comprising the steps of: (a) synthesizing porous microbeads and modifying their surfaces; and (b) attaching ceria nanoparticles to the surfaces of the modified porous microbeads.

[0039] In one exemplary implementation, step (a) may involve modifying the surface of the porous microbeads with a formulation containing at least one functional group selected from the group consisting of amine, thiol, and carboxylic groups.

[0040] In one exemplary implementation, the method may further comprise the step of (c) coating the surface of the porous microbeads to which the ceria nanoparticles are attached with a polymer.

[0041] In an exemplary implementation, step (c) may produce at least one selected from the group consisting of, but not limited to, alginate-coated porous microbeads, PEG (polyethylene glycol)-coated porous microbeads, PVP (polyvinylpyrrolidone)-coated porous microbeads, and combinations thereof.

[0042] The present invention will be described in more detail below with reference to examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and that the scope of the present invention is not limited by these examples. [Example]

[0043] Experimental materials and methods (1-1) Experimental materials Mesitylene, ammonium fluoride, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (Pluronic® P-123) Mn ~ 5,800, hydrochloric acid, nitric acid, alginic acid sodium salt from brown algae, sodium bicarbonate, tetraethyl orthosilicate, 6-aminohexanoic acid, ethyl alcohol, isopropyl alcohol, and acetone were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cerium(III) nitrate hexahydrate was purchased from Alfa Aesar. Methoxy PEG-amide succinimidyl glutarate 5000 was purchased from Sanbio (Anyang, Korea). All reagents were used as received without further purification.

[0044] (1-2) Synthesis of millimeter-sized porous silica microbeads Porous silica microbeads were synthesized using a silica sol-gel method using an organic template. Specifically, (1) 16 g of Pluronic® P-123, 80 mL of HCl, 4 mL of 46 mg / mL ammonium fluoride, and 12 g of TMB were dissolved in 516 mL of deionized water (DI water) and stirred at 50 °C for 4 hours. 36.8 mL of TEOS was then added to the mixture and allowed to react for 20 hours. The synthesized porous silica particles were then subjected to a hydrothermal reaction at 100 °C for 24 hours, after which the resulting porous silica particles were filtered and dried. (2) 10 mL of a 20 mg / mL alginate solution was mixed with 10 mL of 20, 60, or 100 mg / mL porous silica particles, and the mixture was injected into a 100 mM CaCl2 solution using a syringe pump at 12 kV high voltage to synthesize porous silica / alginate microbeads. The silica precursor, prepared by reacting 17 mL of 0.15 M HNO3 with 68 mL of TEOS for 1 hour, was mixed with the porous silica / alginate microbeads and allowed to react for 24 hours. After the reaction, the collected microbeads were washed with IPA, dried, and then calcined at 550 °C for 6 hours to remove the alginate and P123 and generate pores, ultimately yielding porous silica microbeads.

[0045] (1-3) Synthesis of ceria nanoparticles 1.3117 g of 6-aminohexanoic acid and 70 μL of HCl were dissolved in 60 mL of DI water and heated to 95°C. 1.08557 g of cerium(III) nitrate hexahydrate was then added to the mixture and allowed to react for 1 minute. The synthesized ceria nanoparticles were precipitated in 500 mL of acetone, washed by centrifugation at 10,000 rpm for 10 minutes three times, and then dried in a vacuum oven for 24 hours. The dried ceria nanoparticles were redispersed in DI water and stored at 4°C until use.

[0046] (1-4) Preparation of ceria nanoparticles / porous silica microbeads The surface of porous silica microbeads is negatively charged, while ceria nanoparticles are positively charged. This difference in polarity induces electrostatic bonding, allowing the production of ceria nanoparticles / porous silica microbeads. 1 g of porous silica microbeads was mixed with 20 mL of a 5 mg / mL ceria nanoparticle solution and allowed to react for 24 hours. The mixture was then washed three times with deionized water and three times with acetone, then recovered and dried in a vacuum oven for 24 hours. The loading efficiency of the ceria nanoparticles was calculated by measuring the amount of ceria nanoparticles present in the supernatant using ICP-MS and subtracting the amount of ceria nanoparticles from the amount of ceria nanoparticles mixed.

[0047] (1-5) Preparation of PEG-coated ceria nanoparticles / porous silica microbeads To coat porous silica microbeads with polyethylene glycol (PEG), 500 mg of methoxy PEG-amide succinimidyl glutarate 5000 was dissolved in sodium bicarbonate buffer (pH 8.0), to which 1 g of ceria nanoparticles / porous silica microbeads was added and mixed for 24 hours. After the reaction, the PEG-coated ceria nanoparticles / porous silica microbeads were washed three times with deionized water and stored at 4°C until use.

[0048] (1-6) Preparation of PVP-coated ceria nanoparticles / porous silica microbeads One gram of PVP (polyvinylpyrrolidone) was dissolved in deionized water and mixed with 1 gram of ceria nanoparticles / porous silica microbeads for 24 hours. After the reaction, the PVP-coated ceria nanoparticles / porous silica microbeads were washed three times with deionized water and stored in deionized water at 4°C until use.

[0049] (1-7) Characterization of porous silica microbeads The surface morphology and elemental analysis of all microbeads were performed by SEM and EDS (JSM 7000F, JEOL, Japan). The pore size, pore volume, and surface area were measured using the Brunaur-Emmett-Teller (BET) method.

[0050] (1-8) Analysis of the ROS removal ability of microbeads in vitro 10, 25, and 50 mg of porous silica microbeads or ceria nanoparticle / porous silica microbeads were mixed with 500 μM hydrogen peroxide (HO) solution. After 2 hours, 50 μL of the supernatant was collected and mixed with 50 μL of a working solution containing 100 μM AmplexRed reagent and 0.2 U / mL horseradish peroxidase (HRP). The reaction mixture was incubated at room temperature for 30 minutes. The reaction product, resorufin, exhibited fluorescence at an excitation wavelength of 545 nm and an emission wavelength of 590 nm using a microplate reader (Varioskan LUX, Thermo, Massachusetts, USA). The hydroxyl-radical antioxidant capacity of the porous silica microbeads and ceria nanoparticle / porous silica microbeads was measured using the HORAC Activity Assay Kit. 10 mg of porous silica microbeads and ceria nanoparticle / porous silica microbeads were prepared in a 1.5 mL tube. The porous silica microbeads and ceria nanoparticle / porous silica microbeads were mixed with 40 μL of deionized water, 280 μL of fluorescein probe, 40 μL of hydroxyl radial initiator, and 40 μL of Fenton reagent and incubated at room temperature for 60 minutes with gentle shaking. 200 μL of sample solution was withdrawn and measured every 5 minutes for a total of 1 hour at excitation 480 nm and emission 530 nm (Varioskan LUX, Thermo, Massachusetts, USA). All measurements were performed at least in triplicate.

[0051] (1-9) Analysis of in vitro ROS removal ability using microbeads in a cartridge system A cartridge with a volume of approximately 1 mL was prepared, and 8 mm mesh filters were placed at the top and bottom of the cartridge to prevent particle leakage. 200 mg of porous silica microbeads, ceria nanoparticles / porous silica, and PVP-coated ceria nanoparticles / porous silica microbeads were loaded into the prepared cartridge. Using a syringe pump, 2 mM hydrogen peroxide (HO) solution was added to the top at a flow rate of 1 mL / min for 10 minutes. After 10 minutes, 50 μL of the sample was transferred to a 96-well plate, and the residual HO solution was re-injected into the cartridge for 1, 2, 4, and 7 cycles. The hydrogen peroxide removal efficiency was calculated using the same method (Amplex Assay Kit).

[0052] (1-10) Blood adsorption test of microbeads The blood adsorption test was performed by adding 10 mg of porous silica microbeads, ceria nanoparticles / porous silica microbeads, or PVP-coated ceria nanoparticles / porous silica microbeads to 10 mL of blood and incubating at 37°C for 2 hours.

[0053] (1-11) Analysis of the therapeutic effect of microbeads in a cartridge system Sepsis was induced in rats by intravenous injection of 5 mg / kg of endotoxin (Lipopolysaccharide; LPS). Catheters were inserted into the carotid artery, femoral artery, and femoral vein, and a blood perfusion circuit, cartridge, and perfusion pump were connected. For the control group, the cartridge was removed. Ten minutes after sepsis induction, extracorporeal blood circulation was initiated by operating the perfusion pump at a rate of 1.5 mL / min. After sepsis induction, 30 mL / kg of saline was intravenously injected. When mean arterial pressure (MAP) fell below 60 mmHg, the vasopressor norepinephrine was infused at a maximum rate of 1 mcg / kg / min. Extracorporeal blood circulation was stopped after 4 hours. Over a 4-hour period, rats were observed for mortality, mean arterial pressure, and vasopressor use, and these were recorded. In experiments measuring blood lactate levels and leukocyte-released hydrogen peroxide levels, blood samples are taken at 1 hour for comparison to eliminate bias due to individual deaths. [Example]

[0054] Characterization of porous silica microbeads Porous silica microbeads were prepared by synthesizing alginate hydrogel microbeads containing porous silica microparticles and using them as a template (Figure 3). Subsequently, the porous silica microbeads were bonded together via a silica sol-gel reaction in the porous silica / alginate hydrogel. Finally, the porous silica microbeads were obtained by calcination, which removes the organic substances, alginate and P123. Alginate hydrogel microbeads without porous silica particles underwent the same process, resulting in a significant reduction in size and a distorted shape. However, alginate hydrogel microbeads containing porous silica microparticles maintained a similar size after calcination. To control the porosity of the porous silica microbeads, the concentration of porous silica microparticles in the alginate hydrogel was varied (Figure 4). The surface area and pore volume varied depending on the concentration of porous silica microparticles: at 1 wt%, the surface area was 207.33 m, respectively.2 / g, pore volume 0.45 cm 3 / g and 3 wt% respectively have a surface area of ​​244.73 m 2 / g, pore volume 0.56 cm 3 / g, and at 5 wt% the surface area is 395.17 m 2 / g, pore volume 0.84 cm 3 / g. Thus, it was found that the porosity (surface area and pore volume) increased as the concentration of porous silica microparticles increased. In the experiment, high porosity was desirable, but the alginate solution containing 5 wt% porous silica was too viscous and had low productivity, so in future experiments, a 3 wt% concentration of porous silica was used. [Example]

[0055] Loading efficiency of ceria nanoparticles in fabricated ceria nanoparticle / porous silica microbeads Ceria nanoparticles have a positive surface charge and are easily absorbed onto the negatively charged surface of porous silica microbeads (Figures 5 and 6). 1 g of porous silica microbeads was mixed with 5 mg / mL ceria nanoparticles in 20 mL of solution, and the ceria nanoparticles were adsorbed onto the porous silica microbeads. As a result, the ceria nanoparticle / porous silica microbeads mixture showed a slight yellow color due to the loaded ceria nanoparticles (Figure 7). Furthermore, ICP-MS analysis of the supernatant (remaining ceria nanoparticles) after mixing the ceria nanoparticles and porous silica microbeads confirmed 100% loading efficiency. SEM observation revealed that the surface of the porous silica microbeads was not uniform but consisted of a combination of smooth clusters, whereas the surface of the ceria nanoparticle / porous silica microbeads showed a rough texture throughout the clusters. Energy-dispersive X-ray spectroscopy (EDS) confirmed the presence of elemental Ce in the ceria nanoparticle / porous silica microbeads, indicating the adsorption of ceria nanoparticles (Figure 8). [Example]

[0056] Analysis of PVP-coated ceria nanoparticles / porous silica microbeads for hemocompatibility To improve blood compatibility, PVP was coated onto ceria nanoparticles / porous silica microbeads by physical adsorption (Figure 9). One gram of ceria nanoparticles / porous silica microbeads was mixed with 20 mL of a 50 mg / mL PVP aqueous solution and reacted for 24 hours. The resulting PVP-coated ceria nanoparticles / porous silica microbeads were then washed five times with deionized water and collected. The resulting dispersion was then stored in a refrigerator. Thermogravimetric analysis (TGA) confirmed that the PVP coating was approximately 8% by weight on the synthesized PVP-coated ceria nanoparticles / porous silica microbeads (Figure 10). [Example]

[0057] Results of ROS removal confirmation for ceria nanoparticles / porous silica microbeads and PVP-coated ceria nanoparticles / porous silica microbeads Hydrogen peroxide (HO) and hydroxyl radical analysis was performed using porous silica microbeads without ceria nanoparticles, ceria nanoparticle / porous silica microbeads, and PVP-coated ceria nanoparticle / porous silica microbeads. Results showed that the porous silica microbeads without ceria nanoparticles removed almost no HO, whereas both microbeads with ceria nanoparticles had sufficient ROS removal capacity (Figures 11 and 12). This means that the ROS removal capacity was maintained without any decrease even after PVP coating.

[0058] To confirm the amount of ceria nanoparticles required for porous microbeads to have sufficient ROS removal properties, PVP-coated ceria nanoparticle / porous silica microbeads loaded with 2, 4, 10, 20, 40, 100, and 200 mg of ceria nanoparticles per gram of microbeads were used. The microbeads were incubated in 2 mL of 500 μM HO for 1 hour. It was confirmed that increasing the amount of ceria nanoparticles resulted in higher reactive oxygen species (hydrogen peroxide) removal capacity (Figure 13). PVP-coated ceria nanoparticle / porous silica microbeads loaded with 2 mg / g of ceria nanoparticles showed almost no HO removal, whereas PVP-coated ceria nanoparticle / porous silica microbeads loaded with 4 mg / g of ceria nanoparticles showed approximately 30%, 50%, and over 80% removal efficiencies, respectively. When 100 mg or more of ceria nanoparticles were loaded, the reactive oxygen species removal ability was over 90%, and no significant difference in removal ability was observed. Therefore, in future experiments, ceria nanoparticle / porous silica microbeads coated with PVP, which loaded 100 mg of ceria nanoparticles per 1 g of microbeads, were prepared and used to create cartridges. [Example]

[0059] Results of confirming the ROS removal ability of ceria nanoparticles / porous silica microbeads in a hemoperfusion cartridge We reproduced the actual usage environment and confirmed whether the ceria nanoparticles / porous silica microbeads could effectively work within the cartridge and remove ROS.

[0060] Figure 14 shows photographs of each cartridge component fabricated using a 3D printer and the assembled cartridge. A cartridge for removing reactive oxygen species from blood was fabricated by placing 200 mg of porous microbeads inside the cartridge. As examples, cartridges containing porous silica microbeads, ceria nanoparticles / porous silica microbeads, and PVP-coated ceria nanoparticles / porous silica microbeads were fabricated (Figure 15). To test the reactive oxygen species removal ability of each cartridge, perfusion and recovery were repeated with 10 mL of 2 mM HO solution, and the ROS removal efficiency was measured over seven cycles (Figure 16). The results showed that the porous silica microbeads were unable to remove HO, whereas the ceria nanoparticles / porous silica microbeads demonstrated removal efficiencies of approximately 40% after one cycle, approximately 50% after two cycles, and approximately 80% and 95% after four and seven cycles, respectively. Furthermore, despite being coated with PVP polymer, the PVP-coated ceria nanoparticles / porous silica microbeads exhibited a similar ROS scavenging effect to the ceria nanoparticles / porous silica microbeads. In other words, it was confirmed that excellent catalase-mimetic activity was maintained even after PVP coating.

[0061] Unlike the previous experiment, in which the hydrogen peroxide solution that had passed through the cartridge was collected and passed through the cartridge again, we measured the ROS removal efficiency over seven cycles after passing a new hydrogen peroxide solution through the cartridge each time. Even with a high concentration of hydrogen peroxide, the ROS removal rate remained above 60% for the first seven cycles (Figure 17). Furthermore, after the seven repeated experiments, we waited 24 hours and then passed the hydrogen peroxide solution through the cartridge again for the 8th, 9th, 10th, and 11th cycles. After 24 hours, we confirmed that the catalase-mimetic activity was restored even when the hydrogen peroxide solution was passed through the cartridge for 8 to 11 cycles.

[0062] This indicates that ceria nanoparticles / porous silica microbeads and PVP-coated ceria nanoparticles / porous silica microbeads may be capable of removing ROS in future external blood cartridge systems. [Example]

[0063] Comparison of ROS removal ability between porous and non-porous glass microbeads in a hemoperfusion cartridge The catalase-mimetic properties of cartridges fabricated with non-porous glass microbeads and porous PES (polyethersulfone) polymer microbeads loaded with ceria nanoparticles were compared with those of a PVP-coated ceria nanoparticle / porous silica microbead cartridge (Figure 18). While microbeads without ceria nanoparticles were unable to remove hydrogen peroxide, all cartridges fabricated with ceria nanoparticle-loaded microbeads demonstrated hydrogen peroxide removal. However, while non-porous glass microbeads removed a certain level of hydrogen peroxide, their efficiency was lower (approximately 40%) than that of other porous microbeads. When the porous microbead composition was changed to PES polymer, the catalase-mimetic properties remained at approximately 90%, comparable to the 95% level achieved by cartridges based on porous silica microbeads. This indicates that the large amount of ceria nanoparticles loaded on porous microbeads allows them to exhibit superior reactive oxygen species removal capacity compared to non-porous microbeads at the same volume. [Example]

[0064] Hemoperfusion analysis results Red blood cells in the blood are easily hemolyzed or agglutinated by reactions with external substances. To be used in the bloodstream, microbeads for hemoperfusion must not react with or destroy red blood cells. Ten milligrams of porous silica microbeads, ceria nanoparticle / porous silica microbeads, or PVP-coated ceria nanoparticle / porous silica microbeads were mixed with 1 mL of red blood cell solution and allowed to react for two hours. The microbeads were then separated by centrifugation, and the absorbance of the supernatant was measured to analyze hemolyzed red blood cells. None of the microbeads tested exhibited hemolytic properties (Figures 19 and 20).

[0065] To confirm the degree of adsorption of microbeads to red blood cells in blood, 100 mg of porous silica microbeads, ceria nanoparticle / porous silica microbeads, and ceria nanoparticle / porous silica microbeads coated with alginate, PEG, or PVP were incubated with 10 mL of blood at 37°C for 2 hours. After centrifugation and washing three times with PBS, the microbeads were observed (Figure 21). The porous silica microbeads and ceria nanoparticle / porous silica microbeads showed red color due to adsorption of red blood cells, while the alginate and PEG-coated ceria nanoparticle / porous silica microbeads showed relatively low adsorption of red blood cells. However, the PVP-coated ceria nanoparticle / porous silica microbeads showed almost no adsorption of red blood cells, retaining their original color, indicating no blood adsorption. Therefore, PVP appears to be the most suitable polymer coating material for preventing adsorption of blood to microbeads. [Example]

[0066] Evaluation of the therapeutic effect of PVP-coated ceria nanoparticles / porous silica microbeads in a hemoperfusion cartridge When rats (rats) were induced with sepsis by intravenous injection of LPS and underwent extracorporeal blood purification (ECP) (Figure 22), the group that underwent ECP showed a statistically significant decrease in the concentration of hydrogen peroxide released from leukocytes compared to the control group (Figure 23), and blood lactate levels, an indicator of the severity of septic shock, also decreased significantly (Figure 24). The control group had a 100% mortality rate (n=7 of 7), while the group that underwent ECP had a 0% mortality rate (n=0 of 7), demonstrating a significant difference in survival rate (Figure 25). The ECP group also had significantly higher mean arterial pressure (MAP) values ​​(Figure 26), and required less vasopressor medication compared to the control group (Figure 27).

Claims

1. Porous microbeads containing ceria nanoparticles for extracorporeal hemoperfusion.

2. 10. The porous microbeads of claim 1, wherein the porous microbeads scavenge reactive oxygen species through the catalytic action of ceria nanoparticles.

3. 2. The porous microbeads according to claim 1, wherein the size (diameter) of the porous microbeads is 1 to 1500 μm.

4. The pore volume of the porous microbeads is 0.1 to 13.6 cm ３ 2. The porous microbeads of claim 1, wherein the pore size is 1 / g.

5. The surface area of ​​the porous microbeads is 100 to 1,000 m ２ 2. The porous microbeads of claim 1, wherein the pore size is 1 / g.

6. 10. The porous microbeads of claim 1, wherein the porous microbeads comprise 3 mg / g or more of ceria nanoparticles.

7. 2. The porous microbeads according to claim 1, wherein the porous microbeads are at least one selected from the group consisting of porous microbeads coated with alginate, porous microbeads coated with PEG (polyethylene glycol), porous microbeads coated with PVP (polyvinylpyrrolidone), and combinations thereof.

8. 8. The porous microbeads according to claim 7, wherein the porous microbeads are PVP (polyvinylpyrrolidone) coated porous microbeads.

9. An extracorporeal hemoperfusion cartridge comprising the porous microbeads according to any one of claims 1 to 8.

10. 10. The extracorporeal blood perfusion cartridge according to claim 9, wherein the volume ratio of the porous microbeads is 50 to 90% of the entire extracorporeal blood perfusion cartridge.

11. An extracorporeal blood purification device comprising the extracorporeal hemoperfusion cartridge according to claim 9.

12. A method for extracorporeal blood purification, comprising the steps of: (a) contacting blood isolated from a subject with porous microbeads according to any one of claims 1 to 8; (b) using the porous microbeads to remove reactive oxygen species from the blood; (c) recovering the blood from which the reactive oxygen species have been removed.

13. A method for producing porous microbeads for extracorporeal hemoperfusion according to any one of claims 1 to 8, comprising the following steps: (a) synthesizing and surface modifying porous microbeads; (b) attaching ceria nanoparticles to the surface of the modified porous microbeads;

14. 14. The method of claim 13, wherein step (a) involves modifying the surface of the porous microbeads with a formulation containing at least one functional group selected from the group consisting of amine, thiol, and carboxylic groups.

15. 14. The method of claim 13, wherein the method further comprises the step of (c) coating the surface of the porous microbeads having ceria nanoparticles attached thereto with a polymer.

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