Blood perfusion system for removing active oxygen

Abstract

The present specification provides: an in vitro blood perfusion structure comprising ceria nanoparticles and a support supporting the ceria nanoparticles, whereby reactive oxygen species (ROS) which are in the blood and cause inflammation can be effectively removed in vitro; an in vitro blood perfusion cartridge and an in vitro blood purification device which comprise the structure; a method for manufacturing the structure; and an in vitro blood purification method using the in vitro blood purification device.

Description

Active oxygen removal blood perfusion system

[0001] [Cross-reference to related applications]

[0002] This application claims priority to Republic of Korea Patent Application No. 10-2024-0095254, filed July 18, 2024, Republic of Korea Patent Application No. 10-2024-0095255, filed July 18, 2024, and Republic of Korea Patent Application No. 10-2025-0097049, filed July 17, 2025, the entire contents of which are incorporated herein by reference.

[0003]

[0004] The present specification discloses an extracorporeal blood purification device utilizing ceria nanoparticles and various material-based supports capable of carrying ceria nanoparticles, such as porous microbeads, fibers, and membrane-shaped supports, and an extracorporeal blood purification method using the same.

[0005] Meanwhile, this application was supported by the following research and development project.

[0006]

[0007] [Research and development project supporting this invention]

[0008] [Project ID] 2022R1C1C100961012

[0009] [MSRI Project Number] 08-2023-0123

[0010] [Ministry Name] Ministry of Science and ICT

[0011] [Management Agency] National Research Foundation of Korea

[0012] [Project Name] Individual Basic Research (Ministry of Science and ICT)

[0013] [Project Title] Development of an Extracorporeal Blood Purification System for Sepsis Treatment Based on Multifunctional Microbeads

[0014] [Host] Seoul National University Bundang Hospital

[0015] Research Period: March 1, 2022 - February 28, 2025

[0016]

[0017] [Research and development project supporting this invention]

[0018] [Project ID] RS-2023-00222910

[0019] [MSRI Project Number] 08-2023-0191

[0020] [Ministry Name] Ministry of Science and ICT

[0021] [Management Agency] National Research Foundation of Korea

[0022] [Project Name] Bio and Medical Technology Development (R&D)

[0023] [Project Name] Developing Medical Field Application Technologies for Five Major Diseases and Fostering Physician Scientists through a Customized Future Medical Research Center in the Era of 6P Medicine

[0024] [Host] Seoul National University Bundang Hospital

[0025] Research Period: April 1, 2023 - December 31, 2026

[0026]

[0027] [Research and development project supporting this invention]

[0028] [Project ID] 1711189569

[0029] [Detailed Assignment Number] 2022R1A2B5B02002097

[0030] [Ministry Name] Ministry of Science and ICT

[0031] [Management Agency] National Research Foundation of Korea

[0032] [Project Name] Individual Basic Research (Ministry of Science and ICT)

[0033] [Project Title] Development of a Nanoparticle-Based Multiple Sclerosis Treatment Vaccine that Induces Enhanced Antigen-Specific Immune Tolerance

[0034] [Name of the project performing organization] Sungkyunkwan University

[0035] Research Period: March 1, 2023 - February 28, 2025

[0036] Sepsis is a serious infection characterized by a systemic inflammatory response. Sepsis is an exaggerated and misguided response to infection, leading to tissue damage and organ dysfunction, potentially leading to life-threatening complications. The systemic inflammatory response syndrome (SIRS), 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, plays a significant pathophysiological role in sepsis. Various drugs have been developed and clinically tested to treat sepsis, but none have proven effective. Blood purification therapies (e.g., Toraymyxin, Cytosorb), which remove pathogen-associated substances and cytokines externally, have been developed as a novel approach to treating sepsis. These therapies have raised expectations and undergone clinical trials, but have not been successful, and their use is not recommended in clinical guidelines. Reactive oxygen species are a critical factor in SIR syndrome, including sepsis, where excessive production or inadequate removal can damage cells and promote inflammation, triggering tissue damage and organ dysfunction. However, existing blood purification treatments have a clear limitation in that they cannot effectively remove active oxygen.

[0037] Accordingly, the inventors of the present invention, after continuous efforts to develop an effective extracorporeal blood perfusion system, have completed an extracorporeal blood purification device capable of effectively removing reactive oxygen species (ROS) in blood and an extracorporeal blood purification method using the same by employing ceria nanoparticles and further combining or attaching ceria nanoparticles to various materials, especially polymer-based supports.

[0038] One object of the present invention is to provide a structure for extracorporeal blood perfusion using ceria nanoparticles, such as a porous microbead for extracorporeal blood perfusion, a fiber for extracorporeal blood perfusion, or a membrane support for extracorporeal blood perfusion, which can effectively remove reactive oxygen species (ROS) in the blood that cause inflammation and damage from the body.

[0039] Another object of the present invention is to provide a polymer-based extracorporeal blood perfusion structure, such as a porous microbead for extracorporeal blood perfusion, a fiber for extracorporeal blood perfusion, or a membrane support for extracorporeal blood perfusion, which can effectively support ceria nanoparticles and prevent ceria nanoparticles or a fragment of the structure containing the same from falling off and flowing into the blood.

[0040] Another object of the present invention is to provide an extracorporeal blood perfusion cartridge comprising the above structure.

[0041] Another object of the present invention is to provide an extracorporeal blood purification device comprising the above structure.

[0042] Another object of the present invention is to provide a method for manufacturing the above structure.

[0043] Another object of the present invention is to provide an extracorporeal blood purification method using the extracorporeal blood purification device.

[0044] To achieve the above purpose,

[0045] In one aspect, the present invention provides a structure for extracorporeal blood perfusion comprising ceria nanoparticles and a support carrying the ceria nanoparticles.

[0046] In an exemplary embodiment, the support preferably comprises a polymer, and preferably comprises the polymer in an amount of at least 50 wt% of the total weight.

[0047] In an exemplary embodiment, the polymer may be functionalized to support ceria nanoparticles. The support may have a mesoporous or macroporous structure and provide a large surface area for the introduction of a large amount of ceria nanoparticles. The support may be functionalized to induce strong electrostatic bonding with the ceria nanoparticles by introducing a sulfone group, or to form strong covalent bonds with the ceria nanoparticles by introducing an amine group or a carboxyl group. Such physical structure and chemical functionalization allow the ceria nanoparticles to be stably supported in large amounts on the support and effectively prevent them from being eluted in a blood perfusion environment.

[0048] In an exemplary embodiment, the polymer has excellent mechanical strength and biocompatibility in a blood perfusion environment, and has properties that enable it to be functionalized to support ceria nanoparticles. For example, various polymers described below can be used, and preferably, it can include at least one selected from the group consisting of polystyrene (PS), polyethersulfone (PES), and polyacrylonitrile (PAN). Specifically, polystyrene (PS) is particularly advantageous for supporting and surface modifying CeNPs due to its ease of introducing various functional groups, and polyethersulfone (PES) is characterized by inherent hydrophilicity and excellent biocompatibility. Polyacrylonitrile (PAN) provides excellent chemical resistance and a stable structure.

[0049] In an exemplary embodiment, the structure for extracorporeal blood perfusion may be one or more of a porous microbead, a fiber, or a membrane support for extracorporeal blood perfusion.

[0050] In an exemplary embodiment, the membrane support may be a dialysis membrane or an ultrafiltration membrane.

[0051] In an exemplary embodiment, the extracorporeal blood perfusion structure can remove reactive oxygen species by the catalytic action of ceria nanoparticles.

[0052] In an exemplary embodiment, the ceria nanoparticles may be bound, attached or supported on the surface of the support through electrostatic bonding, chemical bonding such as covalent bonding or physical adsorption after surface modification.

[0053] In an exemplary embodiment, the extracorporeal blood perfusion structure may be coated with one or more selected from the group consisting of polyethylene glycol (PEG) and polyvinylpyrrolidone (PVP).

[0054] In another aspect, the present invention provides an extracorporeal blood perfusion cartridge comprising the extracorporeal blood perfusion structure.

[0055] In another aspect, the present invention provides an extracorporeal blood purification device including the extracorporeal blood perfusion cartridge.

[0056] In another aspect, the present invention provides an extracorporeal blood purification method comprising the following steps.

[0057] (a) a step of bringing blood separated from a subject into contact with the extracorporeal blood perfusion structure;

[0058] (b) a step of removing reactive oxygen species from the blood using the structure; and

[0059] (c) A step of recovering blood from which the above reactive oxygen species have been removed.

[0060] In another aspect, the present invention provides a method for manufacturing a structure for extracorporeal blood perfusion, comprising the following steps.

[0061] (a) a step of preparing a support capable of supporting ceria nanoparticles; and

[0062] (b) A step of binding ceria nanoparticles to the above structure.

[0063] In an exemplary embodiment, step (a) may include a step of modifying the surface of the manufactured support.

[0064] In an exemplary embodiment, the method may further include a step of coating a polymer on the surface of the extracorporeal blood perfusion structure to which the ceria nanoparticles are attached to enhance hemocompatibility.

[0065] In one aspect, the present invention provides a method for effectively removing in vitro reactive oxygen species (ROS) in the blood that cause inflammation and damage by attaching ceria nanoparticles to a support capable of supporting ceria nanoparticles, preferably a polymer-based support, more preferably various polymer-based supports that can be functionalized to support ceria nanoparticles, such as porous microbeads, fibers, and membrane supports. Furthermore, by using a polymer-based support, ceria nanoparticles can be effectively supported and ceria nanoparticles or fragments of the structure containing the same can be prevented from falling off from the structure for in vitro blood perfusion and flowing into the blood.

[0066] Furthermore, an extracorporeal blood purification device configured to circulate a patient's blood through an extracorporeal blood perfusion cartridge including such an extracorporeal blood perfusion structure can rapidly and extensively remove reactive oxygen species in both aqueous solutions and actual blood conditions. Therefore, the present invention can be effectively applied to advanced extracorporeal blood perfusion treatment systems.

[0067] In another aspect, the present invention enables continuous removal of reactive oxygen species. To address the difficulty of continuous removal of reactive oxygen species, particularly in extracorporeal perfusion environments, the present invention utilizes a catalyst (ceria nanoparticles) rather than an adsorbent, thereby achieving continuous removal of reactive oxygen species.

[0068] On the other hand, inorganic nanoparticles such as ceria nanoparticles are difficult to develop into drugs because they are difficult to ensure safety when injected into the body, but the present invention has the advantage of ensuring safety because it removes active oxygen by perfusing blood out of the body.

[0069] In another aspect, the present invention can provide the following effects. By maximizing the catalytic properties of ceria nanoparticles, the present invention can contribute to the treatment of sepsis and various inflammatory diseases by quickly and effectively neutralizing excessive reactive oxygen species in the blood. Furthermore, by using a polymer-based soft support compared to a rigid material such as silica, not only can the support of ceria nanoparticles be more effective, but it can also be advantageous in preventing the separation or dissolution of ceria nanoparticles or fragments of the structure containing them from the extracorporeal blood perfusion structure and inflow into the blood. Furthermore, the use of a polymer support provides excellent mechanical properties and durability, allowing for stable performance even during long-term blood perfusion.

[0070] In another aspect, the results of experiments utilizing the extracorporeal blood perfusion structure of the present invention have demonstrated not only the efficacy of removing reactive oxygen species (ROS), but also the stability and therapeutic effect in an actual blood environment, and thus have the advantage of high clinical applicability for sepsis and other inflammatory diseases.

[0071] In another aspect, unlike the existing extracorporeal perfusion treatment that nonspecifically adsorbs all substances that may be helpful to the patient (e.g., anti-inflammatory cytokines), the present invention can selectively remove reactive oxygen species, which are the fundamental problem of the inflammatory-damage response, thereby solving the problems of the existing extracorporeal blood perfusion device.

[0072] In another aspect, the present invention can remove increased reactive oxygen species in severe diseases related to systemic inflammatory response syndrome including sepsis through extracorporeal perfusion, improve severity indicators (e.g., blood pressure, amount of vasopressor used), and improve survival rates.

[0073] In another aspect, the extracorporeal blood perfusion structure of the present invention can be easily integrated into existing blood perfusion devices and cartridge systems, making it easy to apply in various treatment environments.

[0074] In another aspect, the structure for extracorporeal blood perfusion of the present invention can minimize nonspecific protein adsorption and thrombus formation upon contact with blood and suppress biological reactivity by coating the surface with PVP or the like.

[0075] Figures 1 to 3 are results showing the PS / DVB microbead - CeNPs loading results of an embodiment of the present invention, where Figure 1 is a chemical formula showing the introduction of sulfonation, Figure 2 is a photograph showing the sulfonation treatment of PS / DVB microbeads, and Figure 3 is a characteristic peak graph showing the introduction of sulfone groups (-SO3H) in sulfonated PS / DVB microbeads.

[0076] FIGS. 4 to 6 are optical microscope and SEM photographs of microbeads of an embodiment of the present invention, wherein FIG. 4 is a PS / DVB microbead, FIG. 5 is a sulfonated PS / DVB microbead (S-PS / DVB), and FIG. 6 is a sulfonated PS / DVB microbead carrying ceria (S-PS / DVB@Ce).

[0077] Fig. 7 is an EDS element mapping image of an embodiment of the present invention, showing the results of EDS (Energy Dispersive X-ray Spectroscopy) analysis on S-PS / DVB@Ce microbeads.

[0078] Figure 8 is an image of an S-PS / DVB bead with a sulfonated group already introduced (pre-sulfonated) and rich in meso- and macro-pores, observed using a scanning electron microscope (SEM) in an embodiment of the present invention.

[0079] Figure 9 is the EDS element mapping result of the same bead used in Figure 8.

[0080] FIG. 10 is a scanning electron microscope (SEM) image and energy dispersive X-ray spectroscopy (EDS) analysis result of a structure (S-PS fiber@Ce) in which ceria nanoparticles are supported on sulfonated polystyrene fiber (S-PS fiber) in an embodiment of the present invention.

[0081] Figure 11 is an image taken with an optical microscope showing the change in size of polyethersulfone (PES)-based microbeads according to needle gauges manufactured according to an embodiment of the present invention.

[0082] Figure 12 is an optical microscope image showing the change in size by needle gauge of PES / MCF composite microbeads manufactured by mixing Mesocellular Silica Foam (MCF) into PES in an embodiment of the present invention.

[0083] Figure 13 shows the results of quantitative comparison of the average diameters of PES and PES / MCF microbeads manufactured using a 20G needle in an embodiment of the present invention.

[0084] Figure 14 is a schematic diagram showing the loading and characteristic confirmation of ceria nanoparticles on PES-based microbeads according to an embodiment of the present invention.

[0085] Figure 15 is an optical microscope image and a scanning electron microscope (SEM) image of PES microbeads manufactured according to an embodiment of the present invention.

[0086] Figure 16 is an optical microscope and SEM image of PES@Ce microbeads directly loaded with ceria nanoparticles in an embodiment of the present invention.

[0087] Figure 17 is an optical microscope and SEM image of PES / MCF microbeads manufactured including MCF in an embodiment of the present invention.

[0088] FIG. 18 is an optical microscope and SEM image of (PES / MCF)@Ce microbeads manufactured by loading ceria nanoparticles into composite beads including MCF in an embodiment of the present invention.

[0089] Figure 19 shows the results of UV-Vis absorbance and quantitative analysis confirming that ceria nanoparticles (CeNPs) were loaded on PES and PES / MCF microbeads in an embodiment of the present invention.

[0090] Figure 20 is a result of observing the surface of a PES membrane (film) using a scanning electron microscope (SEM) in an embodiment of the present invention.

[0091] Figure 21 is an EDS mapping analysis result of PES-membrane@Ce, which supports ceria nanoparticles on a PES membrane in an embodiment of the present invention.

[0092] Figure 22 is a photograph showing a process of electrospinning polyacrylonitrile (PAN) dissolved in DMF into an ethanol / water mixed solvent in an embodiment of the present invention.

[0093] Figure 23 is a scanning electron microscope (SEM) image of polyacrylonitrile (PAN) microbeads in an embodiment of the present invention.

[0094] Figure 24 shows surface modification of PAN-based microbeads in an embodiment of the present invention.

[0095] Figure 25 is a vibration peak graph that can confirm surface modification in an embodiment of the present invention.

[0096] Figure 26 shows the EDS mapping results of PAN-COOH microbeads carrying ceria nanoparticles in an embodiment of the present invention.

[0097] Figure 27 shows the results of comparing the reactive oxygen species (ROS) removal performance of PS / DVB-based microbeads in an embodiment of the present invention.

[0098] Figure 28 shows an example of the present invention in which ROS removal ability was evaluated by loading ceria nanoparticles onto porous polystyrene fibers (PS fibers) and sulfonated polystyrene fibers (S-PS@Ce fibers).

[0099] Figure 29 shows the results of evaluating the ROS removal efficacy of PES-based microbeads loaded with ceria nanoparticles (CeNPs) in an embodiment of the present invention.

[0100] Figure 30 is a result showing the sustainability of ROS removal when a 0.2 mM hydrogen peroxide (H2O2) solution was repeatedly perfused using a CeNPs-loaded microbead-based cartridge in an embodiment of the present invention.

[0101] Figure 31 shows the results of comparing the ROS removal efficiency of a PES membrane and a PES membrane (PES@Ce) loaded with ceria nanoparticles (CeNPs) in an embodiment of the present invention.

[0102] Figure 32 is a graph showing the results of evaluating the hydrogen peroxide (H2O2) removal performance according to surface modification of PAN-based microbeads and support of ceria nanoparticles (CeNPs) in an embodiment of the present invention.

[0103] Figure 33 shows the results showing that the blood perfusion system of an embodiment of the present invention significantly improves the survival rate in a severe sepsis animal model.

[0104] Figure 34 shows the results showing that the blood perfusion system of an embodiment of the present invention significantly improves hypotension in a severe sepsis animal model.

[0105] Hereinafter, exemplary implementation examples of the present invention will be described in detail.

[0106] In this specification, “porous” means a structure including mesopores (2 to 50 nm) and macropores (greater than 50 nm) for effective support of ceria nanoparticles, and the pores increase the internal or external surface area of ​​the support, thereby expanding the contact area with the nanoparticles. In other words, the porous structure can provide a large surface area to enable ceria nanoparticles to come into contact with as many ROS as possible.

[0107] In exemplary embodiments of the present invention, ceria nanoparticles are used in a structure for extracorporeal blood perfusion. The reason for using ceria nanoparticles in a structure for extracorporeal blood perfusion is that they have a unique redox cycling capability. Cerium (Ce) has an oxidation state of +3 (Ce 3+ ) and +4 (Ce 4+ ) can be freely switched between superoxide, hydrogen peroxide, hydroxyl radical, hypochlorite, etc., and thus can efficiently remove various reactive oxygen species (ROS) such as superoxide, hydrogen peroxide, hydroxyl radical, hypochlorite, etc. In particular, ceria nanoparticles have high reactivity through oxygen vacancies on their surface and can act as an auto-catalytic antioxidant that repeatedly removes ROS. In addition, unlike existing antioxidants, they have the advantage of being able to function stably for a long time without being consumed.

[0108] Accordingly, exemplary embodiments of the present invention relate, in one aspect, to a structure for extracorporeal blood perfusion comprising ceria nanoparticles and a support carrying the ceria nanoparticles.

[0109] In an exemplary embodiment, the support may be a porous microbead.

[0110] In an exemplary embodiment, the support may be a porous fiber.

[0111] In an exemplary embodiment, the support may be a membrane support. The membrane support may be a semipermeable polymer membrane such as a dialysis membrane, a high-flux membrane, or an ultrafiltration membrane, and may have, for example, an average pore size of about 5 to 50 nm or a molecular weight cutoff limit (MWCO) of about 10,000 to 100,000 Da. Furthermore, in an exemplary embodiment, the membrane support may include a hollow fiber membrane or flat plate membrane support used in conventional hemodialysis, high-flux hemodialysis, or continuous renal replacement therapy (CRRT).

[0112] In an exemplary embodiment, the support preferably comprises a polymer. In particular, it is preferred that the support comprises a polymer, and that the polymer is contained in an amount of 50 wt% or more (which may be defined as polymer-based). By using a flexible support comprising a polymer or preferably polymer-based, as opposed to a rigid material such as silica, not only can the support of ceria nanoparticles be more effective, but it can also be advantageous in preventing the ceria nanoparticles or fragments of the structure containing the same from being separated or dissolved from the structure for extracorporeal blood perfusion and flowing into the blood.

[0113] In an exemplary embodiment, the support may be comprised of at least 50 wt%, at least 60 wt%, at least 70 wt%, at least 80 wt%, at least 90 wt%, or solely of the polymer.

[0114] In an exemplary embodiment, the support is polymer-based (i.e., greater than or equal to 50 wt% polymer), and mesocellular silica foam (MCF) may be additionally added.

[0115] In an exemplary embodiment, the polymer may be a polymer that can be functionalized to enable the support of ceria nanoparticles.

[0116] For example, the polymer may be Chitosan, Chitin, Polyethylene, Polyacrylonitrile, Polyvinylidenefluoride, Polysulfone, Polyethersulfone, Polystyrene, Polyvinyl alcohol, Poly-methylmethacrylate, Cellulose, or Cellulose acetate.

[0117] Chitosan, a natural polymer, possesses excellent biocompatibility and antibacterial properties, and can enhance ROS removal. Chitin, a precursor to chitosan, offers biocompatibility and biodegradability, making it useful for various biomedical applications.

[0118] The above polyethylene (PE) has the advantage of excellent chemical stability and durability, and can be mass-produced at low cost.

[0119] The above polyacrylonitrile (PAN) has excellent mechanical strength and chemical resistance, and can provide a stable structure.

[0120] The above polyvinylidene fluoride (PVDF) provides high chemical stability and heat resistance, so it has the advantage of being able to be used stably even under extreme conditions.

[0121] The above polysulfone (PSU) has high strength and heat resistance and can provide excellent chemical stability.

[0122] The above polyethersulfone (PES) has similar properties to PSU and exhibits excellent biocompatibility.

[0123] The above polystyrene (PS) has the advantage of being able to introduce various functional groups, thereby improving its adsorption capacity.

[0124] The above polyvinyl alcohol (PVA) has the advantage of providing excellent biocompatibility and water solubility, and can be processed into various forms.

[0125] The above polymethylmethacrylate (PMMA) can provide high transparency and biocompatibility, making it suitable for medical devices.

[0126] The above cellulose, as a natural material, can provide biocompatibility and environmental friendliness. In addition, the above cellulose acetate has the advantage of being inexpensive in addition to being biocompatible.

[0127] The various polymer materials described above can be used for porous microbeads, porous fibers, and membrane supports. In particular, cellulose acetate, polysulfone (PSU), and polyethersulfone (PES) can be preferably used as membrane supports.

[0128] In an exemplary embodiment, the support may have a mesoporous or macroporous structure and provide a large surface area for introduction of a large amount of ceria nanoparticles.

[0129] In an exemplary embodiment, the support may be functionalized to induce strong electrostatic bonding with the ceria nanoparticles by introducing a functional group capable of electrostatic bonding with the ceria nanoparticles, such as a sulfone group, or to form strong covalent bonds with the ceria nanoparticles by introducing a functional group capable of covalent bonding, such as an amine group or a carboxyl group. Such physical structure and chemical functionalization can stably support a large amount of ceria nanoparticles on the support and effectively prevent the ceria nanoparticles or fragments of the structure containing the ceria nanoparticles from being separated or eluted in a blood perfusion environment.

[0130] In an exemplary embodiment, the polymer may include at least one selected from the group consisting of polystyrene (PS), polyethersulfone (PES), and polyacrylonitrile (PAN), which have excellent mechanical strength and biocompatibility in a blood perfusion environment and have properties that can be functionalized to enable the loading of ceria nanoparticles. As described above, polystyrene (PS) is particularly advantageous for loading and surface modification of CeNPs due to the ease of introducing various functional groups, and polyethersulfone (PES) is characterized by intrinsic hydrophilicity and excellent biocompatibility. Polyacrylonitrile (PAN) can provide excellent chemical resistance and a stable structure.

[0131] In an exemplary embodiment, the extracorporeal blood perfusion structure can remove reactive oxygen species (ROS) through the catalytic action of ceria nanoparticles. That is, the ceria nanoparticles exhibit excellent antioxidant performance, capable of continuously neutralizing reactive oxygen species through autocatalytic action.

[0132] In an exemplary embodiment, the ceria nanoparticles may be bound, attached or supported on the surface of the support through electrostatic bonding, chemical bonding such as covalent bonding or physical adsorption after surface modification.

[0133] In an exemplary embodiment, the extracorporeal blood perfusion structure may be characterized by evenly dispersing ceria nanoparticles throughout the support to maximize the efficiency of removing reactive oxygen species.

[0134] In an exemplary embodiment, the size (diameter) of the porous microbead 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, or 10 to 1000 μm, or 100 to 1000 μm. The size (diameter) of the porous microbeads may be adjusted in consideration of the size of the cartridge and effective blood perfusion. In addition, as a non-limiting example, the size (diameter) of the porous microbeads may be set to be larger than a blood cell, for example, greater than 10 μm.

[0135] In an exemplary embodiment, the reactive oxygen species may be one or more selected from the group consisting of, but is not limited to, a superoxide anion, hydrogen peroxide, and a hydroxyl radical.

[0136] In an exemplary embodiment, the pore volume of the porous microbead is 0.1 cm 3 / g to 13.5 cm 3 / g, preferably 0.1 cm 3 / g to 5.0 cm 3 / g, more preferably 0.5 cm 3 / g to 1.5cm 3 / g, but is not limited thereto. Porous microbeads having the above pore volume can effectively support ceria nanoparticles and remove reactive oxygen species.

[0137] In an exemplary embodiment, the surface area of ​​the porous microbeads is 100 m 2 / g to 1000 m 2 / g, preferably 200 m 2 / g to 900 m 2 / g more preferably 300 m 2 / g to 800 m 2 / g, but is not limited thereto. Porous microbeads having the above surface area can effectively support ceria nanoparticles and remove reactive oxygen species.

[0138] In an exemplary embodiment, the porous fiber may be a polymer-based or polymer-made porous fiber having a diameter of about 10 to 50 μm. For example, the porous fiber may be formed of polystyrene (PS), polypropylene (PP), polycaprolactam (PA-6), polyacrylonitrile (PAN), or a copolymer thereof, and the surface may be chemically treated to introduce highly active functional groups or to support nanoparticles. The fiber may have a large surface area, be porous, and have a porosity of, for example, about 10% or less, for example, about 0.1 to 10%, to ensure durability and flow properties.

[0139] In an exemplary embodiment, the membrane support may be non-porous, but is preferably porous. For example, it may be provided in the form of a hollow fiber membrane or a flat sheet membrane, and may be a semi-permeable polymer membrane for the purpose of removing specific molecules in blood or plasma. The membrane may generally be formed of polysulfone (PS), polyethersulfone (PES), polyvinylidene fluoride (PVDF), polymethyl methacrylate (PMMA), polyacrylonitrile (PAN), a cellulose derivative, or the like. The physical properties of the membrane may include, for example, an outer diameter of about 200 to 350 μm, an inner diameter of about 150 to 250 μm, a wall thickness of about 30 to 100 μm, a pore size of about 5 to 50 nm, and a porosity of about 30 to 80%. Additionally, the molecular weight cutoff limit (MWCO) is approximately 10,000 to 100,000 Da, the ultrafiltration coefficient (UF coefficient) is approximately 5 to 80 mL / h / mmHg / m², and the surface area is 0.2 to 2.5 m², which can be adjusted depending on the therapeutic purpose. In addition, the membrane can be surface modified, such as coating with a hydrophilic polymer (e.g., PVP), introducing heparin, or introducing a functional group for nanoparticle loading, to improve biocompatibility.

[0140] In an exemplary embodiment, the extracorporeal blood perfusion structure may comprise ceria nanoparticles in an amount of 0.5 mg / g or more. Here, 'mg / g' means the amount (mg) of ceria nanoparticles per 1 g of the support.

[0141] Specifically, the extracorporeal blood perfusion structure may contain ceria nanoparticles in an amount of 0.5 mg / g or more, 1 mg / g or more, 2 mg / g or more, 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 less, 500 mg / g or less, 400 mg / g or less, or 300 mg / g or less, but is not limited thereto. For example, an extracorporeal blood perfusion structure in a cartridge containing ceria nanoparticles at 100 mg / g or 200 mg / g can effectively perform extracorporeal blood perfusion and reactive oxygen species removal. In an exemplary embodiment, the extracorporeal blood perfusion structure may be coated with at least one selected from the group consisting of PEG (polyethylene glycol) and PVP (polyvinylpyrrolidone). The coating prevents the reactivity of the extracorporeal blood perfusion structure with blood, thereby inhibiting hemolysis or aggregation, and thus improving the hemocompatibility of the extracorporeal blood perfusion structure. In this respect, an extracorporeal blood perfusion structure coated with PEG (polyethylene glycol) is preferable, an extracorporeal blood perfusion structure coated with PVP (polyvinylpyrrolidone) is preferable, and an extracorporeal blood perfusion structure coated with PVP (polyvinylpyrrolidone) is more preferable.

[0142] In another aspect, the present invention relates to an extracorporeal blood perfusion cartridge comprising the above extracorporeal blood perfusion structure.

[0143] In an exemplary embodiment, the extracorporeal blood perfusion structure may have a volume ratio of 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, relative to the entire extracorporeal blood perfusion cartridge. For example, the extracorporeal blood perfusion structure may have a volume ratio of 50 to 90% relative to the entire extracorporeal blood perfusion cartridge, but is not limited thereto. Extracorporeal blood perfusion and active oxygen removal can be effectively performed at the above volume ratio.

[0144] In another aspect, the present invention relates to an extracorporeal blood purification device including the extracorporeal blood perfusion cartridge.

[0145] In another aspect, the present invention relates to an extracorporeal blood purification method comprising the following steps:

[0146] (a) a step of bringing blood separated from a subject into contact with the extracorporeal blood perfusion structure;

[0147] (b) a step of removing reactive oxygen species from the blood using the extracorporeal blood perfusion structure; and

[0148] (c) A step of recovering blood from which the above reactive oxygen species have been removed.

[0149] In an exemplary embodiment, the step of removing the reactive oxygen species may be performed for 2 to 24 hours, but is not limited thereto, and the extracorporeal blood perfusion structure enables continuous removal of reactive oxygen species by using a catalyst (ceria nanoparticles) rather than an adsorbent. Therefore, continuous removal of reactive oxygen species is possible for the target as long as necessary without time constraints.

[0150] In another aspect, the present invention relates to a method for manufacturing a structure for extracorporeal blood perfusion, comprising the following steps:

[0151] (a) a step of preparing a support comprising at least one polymer selected from the group consisting of polystyrene (PS), polyethersulfone (PES) and polyacrylonitrile (PAN); and

[0152] (b) A step of binding ceria nanoparticles to the support.

[0153] In an exemplary embodiment, step (a) may include a step of modifying the surface of the manufactured support.

[0154] In an exemplary embodiment, step (a) may modify the support surface using a formulation comprising one or more functional groups selected from the group consisting of amine, sulfonic acid, thiol, carboxylic, hydroxyl, or epoxy groups.

[0155] In an exemplary embodiment, the method may further comprise the step of (c) coating a polymer on the surface of the support to which the ceria nanoparticles are attached.

[0156] In an exemplary embodiment, in the step (c), the coating may be performed with one or more selected from the group consisting of PEG (polyethylene glycol) and PVP (polyvinylpyrrolidone), but is not limited thereto.

[0157] In an exemplary embodiment, the method may further comprise, but is not limited to, a step of coating with heparin and / or a heparin derivative.

[0158] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0159]

[0160] [Example]

[0161] 1. Experimental materials and methods

[0162] (1-1) Ceria nanoparticles (CeNPs)

[0163] Ceria nanoparticles (CeNPs) were synthesized by dissolving 6-aminohexanoic acid (6-AHA, 10 mmol) in 60 mL of deionized water (DI water) and heating to 95°C under magnetic stirring. To this heated solution, 70 μL of hydrochloric acid (HCl) and 50 mL of cerium (III) nitrate hexahydrate (2.5 mmol) were added. The mixture was incubated for 1 min. The resulting CeNPs were then precipitated by mixing with an excess of acetone, washed three times with acetone, and dried in a vacuum oven. Finally, the purified CeNPs were dissolved in deionized water, filtered through a 0.2 μm filter, and stored refrigerated until use.

[0164]

[0165] (1-2) Preparation of support material, functionalization, and loading of CeNPs

[0166] Directly sulfonated polystyrene (PS) microbeads and ceria nanoparticles supported

[0167] Macroporous polystyrene-divinylbenzene (PS-DVB)-based porous beads were used as the base material. These beads possess sufficient internal and external surface areas for effective loading of CeNPs and are capable of introducing or modifying surface functional groups.

[0168] Specifically, the representative physicochemical properties of the above beads are as follows:

[0169] Diameter 300 - 800 μm, pore volume 0.3 mL / g, surface area 950 m² / g, average pore diameter of meso / macro / transport pores 220 Å, average pore diameter of micro pores 15 Å. The sulfonation process was performed by immersing the microbeads in a concentrated sulfuric acid (approximately 95-98%) solution at 80°C for 3 hours. Afterwards, residual acid was removed through a sufficient distilled water washing process and dried.

[0170] Afterwards, the ceria nanoparticles (CeNPs) loading was performed as follows.

[0171] 1 g of sulfonated PS-DVB beads were mixed with 40 mL of a 5 mg / mL CeNP aqueous solution and stirred at room temperature for 24 h to load ceria nanoparticles. After loading, the beads were washed five times with deionized water to remove non-adsorbed particles, dispersed in deionized water, and stored in a refrigerator.

[0172]

[0173] Pre-sulfonated polystyrene (PS) microbeads and ceria nanoparticles

[0174] In another implementation, porous beads based on PS-DVB, already incorporating sulfonic acid functional groups, were used. These beads exhibited strong acid cation exchange properties and enabled the loading of ceria nanoparticles without additional surface treatment.

[0175] Specifically, the representative physicochemical properties of the beads are as follows: diameter 300-1200 μm, pore volume 0.3 mL / g, surface area 660 m² / g, average pore diameter of meso / macro / transport pores 650 Å, and average pore diameter of micro pores 15 Å.

[0176] The ceria nanoparticle loading process was performed under the same conditions as above (5 mg / mL CeNP aqueous solution, stirring for 24 hours, washing 5 times, and then refrigerated storage).

[0177]

[0178] Polystyrene (PS) fiber and ceria nanoparticle support

[0179] Commercially available porous polystyrene fibers or polystyrene-divinylbenzene (PS / DVB) fibers were used as the base material. The fibers have sufficient internal or external surface area for effective loading of CeNPs and are capable of introducing or modifying surface functional groups.

[0180] Specifically, the porous fiber may have a pore volume of 0.3 mL / g or more, a surface area of ​​600 m² / g or more, and an average pore diameter of 500 Å or more. The fiber was subjected to a sulfonation process to introduce negatively charged sulfonic groups onto its surface. The sulfonation process was performed by immersing the fiber in a concentrated sulfuric acid (approximately 95-98%) solution at 80°C for 3 hours. Thereafter, residual acid was removed through a sufficient deionized water washing process, and the fiber was dried.

[0181] Afterwards, ceria nanoparticle (CeNP) loading was performed as follows.

[0182] Sulfonated polystyrene fibers were cut to a size of 100 mm x 100 mm, and 40 mL of ceria nanoparticles at a concentration of 5 mg / mL per fiber were mixed and stirred at room temperature for 24 hours to load the ceria nanoparticles. After loading, the fibers were washed five times with deionized water for 30 minutes each to remove unadsorbed ceria nanoparticles, and then dried under vacuum using ethanol to remove moisture. The sulfonated polystyrene fibers loaded with ceria nanoparticles were then stored in the freezer.

[0183]

[0184] Polyethersulfone (PES)-based microbeads and ceria nanoparticles

[0185] (1) PES microbeads and CeNP loading

[0186] PES microbeads were prepared by electrospinning a 10 wt% PES solution (solvent: DMF) into a 1:1 ethanol / water mixture (electrospinning voltage: 7 kV, PES solution flow rate: 0.5 mL / min).

[0187] The manufactured PES beads (10 mL) were mixed with 40 mL of a ceria nanoparticle (CeNP) aqueous solution at a concentration of 5 mg / mL and stirred at room temperature for 24 hours to load CeNP.

[0188] Afterwards, it was washed five times with deionized water, dispersed in deionized water, and stored in the refrigerator until use.

[0189]

[0190] (2) PES / MCF composite microbeads and CeNP support

[0191] To assist in the loading of ceria nanoparticles, 1 g of mesocellular silica foam (MCF) was dispersed in a 10 wt% PES solution (DMF, 20 mL), and then electrospun into a 1:1 ethanol / water mixture to produce PES / MCF composite microbeads (electrospinning voltage: 7 kV, PES solution flow rate: 0.5 mL / min).

[0192] The manufactured PES / MCF beads (10 mL) were mixed with CeNP aqueous solution (5 mg / mL, 40 mL) under the same conditions as above, soaked for 24 hours, washed five times, dispersed in deionized water, and stored in a refrigerator.

[0193]

[0194] Polyethersulfone (PES) dialysis membrane and ceria nanoparticle support

[0195] In this example, a flat membrane support made of polyethersulfone (PES) was used as a semi-permeable polymer membrane. The membrane had a diameter of 25 mm, a pore size of approximately 30 nm, and a thickness of 110 μm, and was purchased from a commercial SEM (Surface Engineered Membrane) membrane product with a flat-sheet structure.

[0196] This membrane is a structure having essentially similar characteristics to a typical hemodialysis membrane in terms of structural shape, pore size, and materials used, and was used as an exemplary model for implementing a dialysis membrane to evaluate the possibility of supporting ceria nanoparticles of the present invention.

[0197] 40 mL of ceria nanoparticles at a concentration of 5 mg / mL were mixed and stirred at room temperature for 24 hours to load the ceria nanoparticles. After loading, the solution was washed five times with deionized water for 30 minutes each time to remove unadsorbed ceria nanoparticles, and then moisture was removed using ethanol and dried under vacuum.

[0198]

[0199] Polyacrylonitrile (PAN) microbeads and ceria nanoparticles

[0200] Porous microbeads were directly fabricated using PAN polymer (e.g., average molecular weight M approximately 150,000). PAN microbeads were fabricated by electrospinning a 3 wt% PAN solution (solvent: DMF) into a 1:1 ethanol / water mixture (electrospinning voltage: 7 kV, PAN solution flow rate: 0.5 mL / min). The fabricated PAN beads (7.5 mL) were dehydrated to modify the negative charge of the carboxyl groups on the surface, and then mixed with 40 mL of 1 M NaOH and stirred for 24 h. They were then washed five times, each time for 30 min, with deionized water. The PAN beads, which have a negative charge on the carboxyl groups, turned orange in color.

[0201] To load ceria nanoparticles onto PAN beads surface-modified with carboxyl groups, the manufactured beads (10 mL) were mixed with 5 mg / mL of ceria nanoparticles (40 mL) and loaded for 24 hours. After washing five times, the beads were dispersed in deionized water and stored in a refrigerator.

[0202]

[0203] (1-3) Coating process (PVP)

[0204] Microbeads, fibers, and membranes loaded with CeNPs were coated with PVP (polyvinylpyrrolidone) to improve blood compatibility and prevent CeNPs from falling off.

[0205] PVP coating

[0206] CeNP-loaded microbeads, fibers, or membranes (e.g., 1 g) were mixed with a 5 mg / mL PVP solution (20 mL) for 6 h, washed with deionized water, and dried in a vacuum oven. The resulting P-Ce-polymer supports (microbeads, fibers, or membranes) were stored refrigerated until use.

[0207]

[0208] (1-4) Blood perfusion cartridge manufacturing and system configuration

[0209] The cartridge case consisted of an upper, barrel, and lower portion. Each portion of the cartridge case was designed using Fusion 360 software (Autodesk, CA, USA) and manufactured using a SLA-based ProJet 7000 3D printer (3D Systems, SC, USA). Accura ClearVue resin was used to visualize the cartridge interior. Silicone O-rings were inserted into the joints between each portion of the cartridge case to prevent leakage. The assembled cartridge case was filled with 200 mg of blood perfusion microbeads to assemble the blood perfusion cartridge.

[0210]

[0211] (1-5) Sepsis animal experiment

[0212] The experimental groups consisted of a P-Ce-PS / DVB (ceria-impregnated and PVP-coated polystyrene-divinylbenzene microbead) treatment group and an untreated group that received only standard care. Male Sprague-Dawley rats, 9-14 weeks of age and weighing 300-450 g, were used. After initial sedation with 4% isoflurane for anesthesia induction, tiletamine / zolazepam (30 mg / kg) and xylazine (10 mg / kg) were injected intramuscularly. Tracheal intubation was then performed using a 16-gauge catheter, and mechanical ventilation was initiated using an Inspira Advanced Safety Ventilator. Sedation was maintained with 0.5%-1% isoflurane connected to a ventilation circuit, and tramadol (500 mcg) was injected subcutaneously for analgesia.

[0213] Under aseptic conditions, 24-gauge catheters were cannulated into the left common carotid artery, right common femoral artery, and left common femoral vein. Blood pressure was monitored throughout the experiment via a pressure transducer and monitor connected to the arterial line. Body temperature was monitored with a rectal probe and maintained at 36.5°C–37.5°C with an infrared heater. All fluids and medications were administered through a three-way stopcock connected to the left femoral vein catheter. The blood perfusion device circuit began at the left common carotid artery cannula, was connected via tubing to a blood perfusion cartridge, then via additional tubing to a three-way stopcock for infusion and sampling, and was finally connected to the left common femoral vein catheter. The circuit was initially filled with 100 mL of heparinized saline (50 IU / mL) and then flushed with 100 mL of saline. To induce refractory septic shock, 5 mg / kg of LPS derived from E. coli O111:B4 was administered intravenously over 10 minutes. This was followed by fluid resuscitation with 30 mL / kg of normal saline over 10 minutes. Perfusion was initiated using a peristaltic pump, with blood drawn from the left common carotid artery cannula at a flow rate of 5 mL / kg / min. The blood was perfused through a cartridge and returned to the left common femoral vein. When the mean arterial pressure (MAP) was less than 60 mmHg, norepinephrine infusion was initiated at 0.1 mcg / kg / min, with an adjustable rate up to 2 mcg / kg / min depending on the MAP.

[0214] Differences in survival curves between groups were assessed using the log-rank (Mantel-Cox) test. Changes in blood pressure over time between groups were analyzed using a mixed-effects model that considered both fixed effects (treatment group) and random effects (individual variation).

[0215]

[0216] 2. Experimental Results

[0217] (2-1) Characteristic analysis results of manufactured microbeads, fibers, and membranes

[0218] Figures 1 to 3 are results showing the PS / DVB microbead - CeNPs loading results of an embodiment of the present invention, where Figure 1 is a chemical formula showing the introduction of sulfonation, Figure 2 is a photograph showing the sulfonation treatment of PS / DVB microbeads, and Figure 3 is a characteristic peak graph showing the introduction of sulfone groups (-SO3H) in sulfonated PS / DVB microbeads.

[0219] As can be seen in Fig. 3, the FTIR spectrum analysis results show that the characteristic peak (S=O asymmetric / symmetric stretching vibration, 1000 cm) shows the introduction of sulfonic acid groups (-SO3H) in the sulfonated PS / DVB microbeads. -1 ~ 1200 cm -1 It was confirmed that the surface functionalization (sulfonation) of PS / DVB microbeads was successfully achieved. This proves that the surface functionalization (sulfonation) of PS / DVB microbeads was effectively achieved.

[0220] Figures 4 to 6 are optical microscope and SEM photographs of microbeads of examples of the present invention, wherein Figure 4 shows PS / DVB microbeads, Figure 5 shows sulfonated PS / DVB microbeads (S-PS / DVB), and Figure 6 shows sulfonated PS / DVB microbeads loaded with ceria (S-PS / DVB@Ce). Even after sulfonation and loading of ceria nanoparticles, the uniform spherical shape of the microbeads is maintained, showing that ceria nanoparticles are evenly attached to the surface.

[0221] Fig. 7 is an EDS element mapping image of an embodiment of the present invention, showing the results of EDS (Energy Dispersive X-ray Spectroscopy) analysis on S-PS / DVB@Ce microbeads. As can be seen in Fig. 7, sulfur (S) element mapping (left) confirmed that sulfur was widely distributed on the surface of the sulfonated polymer. Cerium (Ce) element mapping (middle) clearly confirmed that ceria nanoparticles (Ce) were effectively supported and evenly distributed on the surface of the microbeads. This strongly proves that sulfonation was successful, and that positively charged CeNPs were stably attached to the introduced sulfonic acid functional groups through electrostatic attraction.

[0222] Figure 8 is an image of S-PS / DVB beads with sulfonated groups already introduced (pre-sulfonated) and rich in meso- and macro-pores, observed using a scanning electron microscope (SEM).

[0223] Surface (left): Beads with a diameter of 300-1200 μm maintain a smooth appearance, while fine irregularities and porous patterns are observed.

[0224] Cross-section (right): A thick, interwoven internal skeleton and a continuous meso- and macropore network are observed, reflecting the bead characteristics of an enlarged average pore diameter of approximately 650 Å. This structure enhances the penetration and loading capacity of ceria nanoparticles (CeNPs).

[0225] Figure 9 is the EDS element mapping result of the same bead used in Figure 8.

[0226] As can be seen in Figure 9, the sulfur (S) signal is uniformly distributed throughout the fiber skeleton, showing that the sulfonic acid group-SO₃H already exists throughout the bead.

[0227] Furthermore, the cerium (Ce) signal was uniformly present throughout the interior, demonstrating that the ceria nanoparticles were stably loaded onto the entire surface and within the pores of the beads. The simultaneous distribution of sulfur and cerium strongly suggests that the nanoparticles were completely immobilized due to electrostatic interactions between the negatively charged sulfonic groups and the positively charged CeNPs.

[0228] Figure 10 shows a scanning electron microscope (SEM) image and energy-dispersive X-ray spectroscopy (EDS) analysis results of a structure (S-PS fiber@Ce) in which ceria nanoparticles are loaded on sulfonated polystyrene fibers (S-PS fiber). The SEM image confirms that the fiber structure is well formed, and the EDS mapping results confirm that ceria (Ce) is uniformly distributed across the fiber surface. In addition, the presence of S element supports the introduction of sulfone groups, and the distribution of Ce element supports ceria loading, respectively.

[0229] Figure 11 is an optical microscope image showing the size changes of polyethersulfone (PES)-based microbeads manufactured according to an embodiment of the present invention according to needle gauge. As the needle gauge was changed to 22G, 20G, and 18G, the average diameter of the microbeads gradually increased, and in particular, the diameter was the largest at approximately 600-800 μm when an 18G needle was used. Under each condition, the microbeads maintained a uniform sphericity.

[0230] Figure 12 is an optical microscope image showing the size change of PES / MCF composite microbeads manufactured by mixing PES with Mesocellular Silica Foam (MCF) according to the needle gauge in an embodiment of the present invention. Similar to PES single polymer beads, the microbeads tended to increase in size as the needle gauge increased, and overall, excellent morphological stability and sphericity were maintained.

[0231] Figure 13 quantitatively compares the average diameters of PES and PES / MCF microbeads manufactured using a 20G needle. PES / MCF composite microbeads exhibited a significantly smaller average diameter than PES single polymer beads (****, p < 0.0001), demonstrating that variations in the composition of the polymer composite can influence the physical behavior of the microbead formation process.

[0232] Figure 14 is a schematic diagram illustrating the loading and characterization of ceria nanoparticles on PES-based microbeads according to an embodiment of the present invention. The first schematic diagram on the left shows basic PES microbeads manufactured by electrospinning a 10 wt% PES solution (DMF) into a 50% ethanol / water mixture. The second schematic diagram illustrates a structure (PES@Ce) in which ceria nanoparticles are physically adsorbed on the surface of PES microbeads immersed in a CeNP aqueous solution. The third schematic diagram shows composite microbeads (PES / MCF) manufactured by electrospinning a mesocellular silica foam (MCF) mixed with a PES solution, and the fourth schematic diagram shows a (PES / MCF)@Ce structure manufactured by adsorbing CeNPs onto the composite beads. MCF contains a porous silica structure inside, which plays a role in increasing the loading capacity of CeNPs.

[0233] Figure 15 shows optical microscope images and scanning electron microscope (SEM) images of PES microbeads manufactured according to an embodiment of the present invention. As confirmed in the optical images, the microbeads manufactured from a single PES polymer maintained a uniform and precise spherical shape, with an average diameter of approximately 600 μm. SEM analysis revealed that the outer surface was smooth, while the inner cross-section had a spongy porous structure, with nano-sized pores uniformly distributed. These microbeads were used as a control group for future loading experiments.

[0234] Figure 16 shows optical microscope and SEM images of PES@Ce microbeads directly loaded with ceria nanoparticles in an embodiment of the present invention. The optical microscope image confirms that the microbeads maintain a spherical structure similar to that of a single PES bead, and SEM analysis reveals that ceria nanoparticles are finely attached to the surface. Furthermore, the nanoporous structure is maintained in the internal cross-section, suggesting that the ceria is evenly dispersed within the matrix without aggregation.

[0235] Figure 17 shows optical microscope and SEM images of PES / MCF microbeads manufactured by incorporating MCF in an embodiment of the present invention. The optical microscope image shows a sphericity and size similar to that of a single PES polymer bead, while SEM analysis confirms that a denser and more complex porous structure is formed due to the introduction of MCF within the PES matrix. This can simultaneously increase the surface area of ​​the beads and strengthen their structure.

[0236] Figure 18 shows optical microscope and SEM images of (PES / MCF)@Ce microbeads manufactured by loading ceria nanoparticles onto composite beads including MCF in an embodiment of the present invention. The optical microscope images confirm excellent morphological stability, and the SEM analysis results show that ceria particles are uniformly attached to the surface, and the internal cross-section confirms that ceria is effectively loaded within the MCF structure, maintaining a porous channel structure while simultaneously presenting ceria in an integrated form within the network structure.

[0237] Figure 19 shows the results of UV-Vis absorbance and quantitative analysis confirming that ceria nanoparticles (CeNPs) were loaded onto PES and PES / MCF microbeads in an embodiment of the present invention. In the left panel, the absorption peak characteristic of CeNPs (around 260 nm) was observed in both PES and PES / MCF microbeads, indicating that CeNPs were successfully loaded. The quantitative analysis results in the right panel confirmed that PES / MCF microbeads loaded more CeNPs than PES at the same volume (1 mL).

[0238] Figure 20 shows the surface of a PES membrane observed using a scanning electron microscope (SEM) in an embodiment of the present invention. The finely textured structure and porous surface identified in the SEM image demonstrate the unique physical properties of the membrane, which are interpreted as a suitable substrate for surface modification and functionalization for supporting ceria nanoparticles (CeNPs).

[0239] Figure 21 shows the results of EDS mapping analysis of PES-membrane@Ce, a PES membrane loaded with ceria nanoparticles, in an embodiment of the present invention. The analysis results confirmed that the Ce signal was uniformly distributed throughout the membrane, qualitatively suggesting that ceria nanoparticles were effectively loaded on the surface. These characteristics support the possibility that the membrane of this embodiment can serve as a foundation for implementing a reactive oxygen species (ROS) removal function.

[0240] Figure 22 shows, in an embodiment of the present invention, the process of electrospinning polyacrylonitrile (PAN) dissolved in DMF into an ethanol / water mixed solvent, in which internal DMF is rapidly removed through solvent exchange with an external solvent, and insoluble PAN is precipitated in the form of beads. As a result, it can be confirmed that white PAN beads are formed.

[0241] Figure 23 is a scanning electron microscope (SEM) image of polyacrylonitrile (PAN) microbeads in an embodiment of the present invention, showing the surface and cross-sectional structures of the microbeads, respectively. In the surface SEM image on the left, the PAN microbeads maintain an overall uniform spherical shape, and in the high-magnification image, a fine porous surface structure is observed. In the cross-sectional image on the right, a porous channel structure unevenly distributed throughout the interior is confirmed, indicating that pores are also formed inside the microbeads.

[0242] These surface and internal structures can serve as a favorable foundation for implementing the function of removing reactive oxygen species (ROS) during extracorporeal blood perfusion by providing a high specific surface area and diffusion path for effective loading of functional nanomaterials such as ceria nanoparticles (CeNPs).

[0243] Figure 24 illustrates the surface modification of PAN-based microbeads in an embodiment of the present invention. PAN is a polymer that originally contains nitrile groups (-CN), and modification into a negatively charged surface is necessary for the support of ceria nanoparticles (CeNPs). Accordingly, PAN microbeads were treated with an alkaline aqueous solution to hydrolyze the nitrile groups and convert them to carboxyl groups (-COOH), which increased the negative charge on the microbead surface. After the treatment, the color of the microbeads changed from white to orange, which was visually confirmed to indicate successful surface modification.

[0244] Figure 25 shows that the existing PAN has a 2243 cm peak due to the nitrile functional group. -1 It can be confirmed that a vibration peak appears at , and in the surface-modified PAN-COOH, due to the OH vibration absorption of the carboxyl group, a vibration peak of 3354 cm -1 A broad peak can be seen at 1664 cm, which is typical of the carboxyl group's C=O. -1 , and negatively charged COO -Symmetric and asymmetric peaks at 1405 and 1564 cm -1 It was confirmed that the surface was modified with carboxyl groups by checking that it was floating on the surface.

[0245] Figure 26 shows the EDS mapping results of PAN-COOH microbeads loaded with ceria nanoparticles in an embodiment of the present invention. Signals of nitrogen (N) and cerium (Ce) elements were distributed across the surface of the microbeads, suggesting that ceria nanoparticles were effectively loaded.

[0246] Figure 27 shows the results comparing the reactive oxygen species (ROS) removal performance of PS / DVB-based microbeads in an embodiment of the present invention. The control group and PS / DVB microbeads without ceria had almost no H2O2 removal effect, but the ceria-loaded S-PS / DVB@Ce microbeads significantly reduced the H2O2 concentration. In particular, excellent ROS removal ability was confirmed in both direct sulfonated and pre-sulfonated methods, indicating that CeNPs were effectively functionalized under both conditions.

[0247] Figure 28 shows an example of the present invention in which the ROS removal ability of porous polystyrene fibers (PS fibers) and sulfonated polystyrene fibers loaded with ceria nanoparticles (S-PS@Ce fibers) was evaluated. Polystyrene fibers not loaded with ceria nanoparticles showed almost no ROS removal efficiency, but sulfonated polystyrene fibers loaded with ceria showed a removal efficiency of approximately 90 percent.

[0248] Figure 29 shows the results of evaluating the ROS removal efficacy of PES-based microbeads loaded with ceria nanoparticles (CeNPs) in an embodiment of the present invention. As can be seen in Figure 29, the non-ceria-loaded control groups (PES and PES / MCF) showed almost no ROS removal effect, whereas the ceria-loaded PES@Ce and (PES / MCF)@Ce microbeads showed significant ROS removal ability, removing about 80% of hydrogen peroxide (H2O2).

[0249] Figure 30 shows the sustainability of ROS removal when a 0.2 mM hydrogen peroxide (H2O2) solution was repeatedly perfused using a CeNPs-loaded microbead-based cartridge in an embodiment of the present invention. 10 mL of H2O2 solution was repeatedly perfused at 1 mL / min through a cartridge filled with 1 mL of microbeads, and the residual H2O2 concentration at each cycle (0, 1, 3, 5, 7) was measured. As a result, both PES@Ce and PES / MCF@Ce microbeads maintained the ability to stably remove H2O2 even after repeated perfusion. In particular, PES / MCF@Ce showed a slightly lower residual H2O2 concentration overall, indicating slightly superior ROS removal efficiency. This result demonstrates that ceria nanoparticles act catalytically to maintain ROS removal activity even after repeated use.

[0250] Figure 31 shows the results of comparing the ROS removal efficiency of a PES membrane and a PES membrane (PES@Ce) loaded with ceria nanoparticles (CeNPs) in an embodiment of the present invention.

[0251] While the PES membrane without ceria impregnation showed little hydrogen peroxide (H2O2) removal ability, the PES@Ce membrane exhibited excellent ROS removal efficiency, removing approximately 95% of H2O2. This suggests that the porous structure of the PES membrane enables effective loading of CeNPs and that the ceria-impregnated membrane can be useful as a material for ROS removal.

[0252] Figure 32 is a graph showing the results of evaluating the hydrogen peroxide (H2O2) removal performance according to surface modification of PAN-based microbeads and support of ceria nanoparticles (CeNPs) in an embodiment of the present invention.

[0253] Here, the experimental group consists of four conditions, each of which is divided as follows.

[0254] (1) PAN: Pure PAN beads without modification or support;

[0255] (2) PAN@Ce: CeNP is directly loaded onto PAN beads,

[0256] (3) PAN-COOH: PAN beads with the surface modified with carboxyl groups (-COOH).

[0257] (4) (PAN-COOH)@Ce: (CeNP loaded on PAN beads modified with carboxyl groups).

[0258] Figure 32 demonstrates that the loading of CeNPs contributes to the removal of H2O2, and that the H2O2 removal ability is maximized, particularly when the PAN surface is modified with carboxyl groups and then loaded with CeNPs. This demonstrates that the modification of carboxyl groups induces stable loading of CeNPs, thereby significantly enhancing the efficiency of removing reactive oxygen species.

[0259] Figure 33 shows that the blood perfusion system of an embodiment of the present invention significantly improves survival rate in a severe sepsis animal model.

[0260] In this experiment, microbeads (PS-PS-DVB@Ce), which were made by loading ceria nanoparticles (CeNPs) onto sulfonated polystyrene-dimethylsulfonyl sulfonate (PS-DVB) and coating their surfaces with PVP, were used for blood perfusion. The group treated with this system showed a 100% survival rate, which was significantly different from the 0% survival rate in the control group that received only standard treatment. These results clearly demonstrate that the S-PS-DVB@Ce-based blood perfusion system of the present invention can effectively reduce mortality due to sepsis.

[0261] Figure 34 shows that the blood perfusion system of an embodiment of the present invention significantly improves hypotension in a severe sepsis animal model. The X-axis represents the time elapsed after LPS administration, and the Y-axis represents the mean arterial pressure (MAP, mmHg). The control group (group receiving only standard treatment, dotted line) showed a persistent decrease in mean arterial pressure, which is a typical pattern of refractory septic shock, whereas the group treated with the blood perfusion system of an embodiment of the present invention (e.g., perfusion using PVP-coated S-PS / DVB@Ce microbeads, solid line) showed a tendency for MAP to be maintained at a constant level or increase, demonstrating that the hypotension condition was effectively improved.

[0262] The present invention relates to an extracorporeal blood perfusion system that can contribute to the treatment of sepsis and various inflammatory diseases by effectively removing reactive oxygen species (ROS) in the blood that cause inflammation from outside the body.

Claims

A structure for extracorporeal blood perfusion, comprising ceria nanoparticles and a support carrying the ceria nanoparticles. In the first paragraph, A structure for extracorporeal blood perfusion, wherein the support is a polymer-based support in which the polymer accounts for 50% or more by weight of the total weight of the support. In the first paragraph, A structure for extracorporeal blood perfusion, wherein the support comprises at least one polymer selected from the group consisting of Chitosan, Chitin, Polyethylene, Polyacrylonitrile, Polyvinylidenefluoride, Polysulfone, Polyethersulfone, Polystyrene, Polyvinyl alcohol, Poly-methylmethacrylate, Cellulose, or Cellulose acetate. In the first paragraph, A structure for extracorporeal blood perfusion, wherein the support comprises at least one polymer selected from the group consisting of polystyrene (PS), polyethersulfone (PES), and polyacrylonitrile (PAN). In the first paragraph, A structure for extracorporeal blood perfusion, wherein the support is a porous microbead. In the first paragraph, A structure for extracorporeal blood perfusion, wherein the support is a porous fiber. In the first paragraph, The above support is a membrane support, a structure for extracorporeal blood perfusion. In the first paragraph, The above extracorporeal blood perfusion structure is an extracorporeal blood perfusion structure that removes reactive oxygen species through the catalytic action of ceria nanoparticles. In paragraph 5, A structure for extracorporeal blood perfusion, wherein the size (diameter) of the above porous microbeads is 1 to 1500 μm. In paragraph 5, The pore volume of the above porous microbeads is 0.1 cm 3 / g to 13.6 cm 3 / g, a structure for extracorporeal blood perfusion. In paragraph 5, The surface area of ​​the above porous microbeads is 100 m 2 / g to 1,000m 2 / g, a structure for extracorporeal blood perfusion. In paragraph 6, The above porous fiber is a structure for extracorporeal blood perfusion, having a diameter of 10 to 50 μm. In paragraph 6, A structure for extracorporeal blood perfusion, wherein the above porous fiber has a porosity of 10% or less. In paragraph 7, A structure for extracorporeal blood perfusion, wherein the membrane support has an outer diameter of 200 to 350 μm, an inner diameter of 150 to 250 μm, a wall thickness of 30 to 100 μm, a pore size of 5 to 50 nm, and a porosity of 30 to 80%. In paragraph 7, The above membrane support is a structure for extracorporeal blood perfusion, having a molecular weight cutoff limit (MWCO) of 10,000 to 100,000 Da, an ultrafiltration (UF) coefficient of 5 to 80 mL / h / mmHg / m², and a surface area of ​​0.2 to 2.5 m². In the first paragraph, The above structure for extracorporeal blood perfusion is a structure for extracorporeal blood perfusion comprising 0.5 mg / g or more of ceria nanoparticles. In the first paragraph, The above structure for extracorporeal blood perfusion is a structure for extracorporeal blood perfusion, which is coated with at least one selected from the group consisting of PEG (polyethylene glycol) and PVP (polyvinylpyrrolidone). An extracorporeal blood perfusion cartridge comprising a structure for extracorporeal blood perfusion according to any one of claims 1 to 17. In paragraph 18, An extracorporeal blood perfusion cartridge, wherein the structure for extracorporeal blood perfusion has a volume ratio of 50% to 90% with respect to the entire extracorporeal blood perfusion cartridge. An extracorporeal blood purification device comprising an extracorporeal blood perfusion cartridge of claim 18. Extracorporeal blood purification method comprising the following steps: (a) a step of contacting blood separated from a subject with an extracorporeal blood perfusion structure according to any one of claims 1 to 17; (b) a step of removing reactive oxygen species from the blood using the extracorporeal blood perfusion structure; and (c) A step of recovering blood from which the above reactive oxygen species have been removed. A method for manufacturing a structure for extracorporeal blood perfusion according to any one of claims 1 to 17, comprising the following steps: (a) a step of manufacturing a structure for extracorporeal blood perfusion comprising one or more polymers; (b) A step of binding ceria nanoparticles to the above extracorporeal blood perfusion structure. In paragraph 22, A method comprising the step (a) of modifying the surface of a structure for extracorporeal blood perfusion using a preparation containing one or more functional groups selected from the group consisting of an amine, sulfonic acid, thiol, carboxylic, hydroxyl, or epoxy group. In paragraph 22, The method further comprises a step of coating the surface of the structure for extracorporeal blood perfusion with at least one polymer selected from the group consisting of PEG (polyethylene glycol) and PVP (polyvinylpyrrolidone).

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