Blood perfusion system for scavenging reactive oxygen species

Abstract

This specification provides a structure for in vitro blood perfusion that can effectively remove reactive oxygen species (ROS) that cause inflammation in the blood, an in vitro blood perfusion tube including the structure and an in vitro blood purification device, a method for preparing the structure, and an in vitro blood purification method using the in vitro blood purification device, wherein the structure for in vitro blood perfusion includes cerium dioxide nanoparticles and a carrier loaded with cerium dioxide nanoparticles.

Description

Cross-reference to related applications

[0001] This application claims priority to Korean Patent Application No. 10-2024-0095254, filed on July 18, 2024; Korean Patent Application No. 10-2024-0095255, filed on July 18, 2024; and Korean Patent Application No. 10-2025-0097049, filed on July 17, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This specification discloses an extracorporeal blood purification device utilizing cerium dioxide nanoparticles and carriers of various material substrates capable of supporting cerium dioxide nanoparticles (such as carriers in the form of porous microspheres, fibers, or membranes), as well as an extracorporeal blood purification method using the extracorporeal blood purification device.

[0003] Meanwhile, this application was supported by the following research and development projects.

[0004] [Support the research and development projects of this invention] [Project Unique Number] 2022R1C1C100961012 [MSRI Project Number] 08-2023-0123 [Department Name] Ministry of Science, Technology and Information [Management Professional Organization] Korea National Research Foundation [Project Name] Personal Basic Research (Department of Science, Information and Communication Technology) [Project Title] Development of an Extracorporeal Blood Purification System for Sepsis Treatment Based on Multifunctional Microbeads [Supervising Institution] Seoul National University Bundang Hospital [Research Period] March 1, 2022 ~ February 28, 2025 [Support the research and development projects of this invention] [Project Unique Number] RS-2023-00222910 [MSRI Project Number] 08-2023-0191 [Department Name] Ministry of Science, Technology and Information [Management Professional Organization] Korea National Research Foundation [Project Name] Biotechnology and Medical Technology Development (R&D) [Project Name] Developing on-site medical application technologies for five major diseases and training medical scientists through a future medical research center that caters to the "6P" medical era. [Supervising Institution] Seoul National University Bundang Hospital [Research Period] April 1, 2023 ~ December 31, 2026 [Support the research and development projects of this invention] [Project Unique Number] 1711189569 [Specific Project Number] 2022R1A2B5B02002097 [Department Name] Ministry of Science, Technology and Information [Management Professional Organization] Korea National Research Foundation [Project Name] Personal Basic Research (Department of Science, Information and Communication Technology) [Project Title] Development of Nanoparticle-Based Vaccines for the Treatment of Multiple Sclerosis that Induce Enhanced Antigen-Specific Immune Tolerance [Name of the Institution Implementing the Project] Sungkyunkwan University [Research Period] March 1, 2023 ~ February 28, 2025 Background Technology Sepsis is essentially a severe infectious state accompanied by a systemic inflammatory response. Sepsis is an excessive and dysregulated response of the body to infection, leading to tissue damage, organ dysfunction, and ultimately, a life-threatening condition. Systemic inflammatory response syndrome (SIRS) is a systemic inflammatory response exhibited by the body in response to various forms of severe stress or injury, such as infection, trauma, burns, and pancreatitis, and it also plays an important pathophysiological role in sepsis. To date, various drugs have been developed and clinical trials conducted for the treatment of sepsis, but their effectiveness has not been verified. As a novel approach to treating sepsis, blood purification therapy (e.g., Toraymyxin, Cytosorb, etc.) to remove pathogen-related substances or cytokines in vitro has been developed and held in high regard, but clinical trials have failed to achieve success, and its use is not recommended in treatment guidelines. In SIRS, including sepsis, excessive production or insufficient clearance of reactive oxygen species (ROS) damages cells and promotes inflammatory responses, which are important factors leading to tissue damage and organ dysfunction. However, traditional blood purification therapies have the significant limitation of not being able to effectively remove ROS.

[0005] Therefore, in order to develop an effective extracorporeal blood perfusion system, through the continuous efforts of the inventors, an extracorporeal blood purification device and an extracorporeal blood purification method using the extracorporeal blood purification device have been realized. The extracorporeal blood purification device can use cerium dioxide nanoparticles, and effectively remove reactive oxygen species (ROS) from the blood by further combining or attaching the cerium dioxide nanoparticles to a carrier of various materials (especially polymers). Summary of the Invention

[0006] Technical problems to be solved One object of the present invention is to provide a structure for in vitro blood perfusion (such as porous microbeads for in vitro blood perfusion, fibers for in vitro blood perfusion, and membrane carriers for in vitro blood perfusion) utilizing cerium dioxide nanoparticles, which can effectively remove reactive oxygen species (ROS) from the blood in vitro that cause inflammation and damage.

[0007] Another object of the present invention is to provide a structure that can effectively load cerium dioxide nanoparticles and prevent cerium dioxide nanoparticles or structural fragments containing them from detaching from a structure for in vitro hemoperfusion and flowing into the blood, particularly polymer-based structures for in vitro hemoperfusion (such as porous microbeads for in vitro hemoperfusion, fibers for in vitro hemoperfusion, and membrane carriers for in vitro hemoperfusion).

[0008] Another object of the present invention is to provide an extracorporeal blood perfusion tube including the structure described above.

[0009] Another object of the present invention is to provide an extracorporeal blood purification device including the structure described above.

[0010] Another object of the present invention is to provide a method for preparing the structure.

[0011] Another object of the present invention is to provide an extracorporeal blood purification method using the aforementioned extracorporeal blood purification device.

[0012] Solution to the problem In order to achieve the aforementioned objective, One aspect of the present invention provides a structure for in vitro blood perfusion, comprising cerium dioxide nanoparticles and a carrier loading the cerium dioxide nanoparticles.

[0013] In one exemplary embodiment, the carrier preferably comprises a polymer, and preferably comprises a polymer comprising more than 50 weight percent (wt%) of the total weight.

[0014] In one exemplary embodiment, the polymer may be a polymer that can be functionalized to support cerium dioxide nanoparticles. The support has a mesoporous or macroporous structure, providing a large surface area to accommodate a large quantity of cerium dioxide nanoparticles. The support may be functionalized by introducing sulfone groups to induce strong electrostatic bonding with the cerium dioxide nanoparticles, or by introducing amine or carboxyl groups to form strong covalent bonds with the cerium dioxide nanoparticles. Such physical structure and chemical functionalization allow for the stable and abundant loading of cerium dioxide nanoparticles onto the support, effectively preventing their elution in a blood perfusion environment.

[0015] In one exemplary embodiment, the polymer exhibits excellent mechanical strength and biocompatibility under blood perfusion conditions, and has the property of being functionalized to achieve loading of cerium dioxide nanoparticles. Various polymers described below can be used, preferably including one or more polymers selected from the group consisting of polystyrene (PS), polyethersulfone (PES), and polyacrylonitrile (PAN). Specifically, polystyrene (PS) readily incorporates various functional groups, which is particularly beneficial for loading and surface modification of CeNPs, while polyethersulfone (PES) is characterized by its inherent hydrophilicity and excellent biocompatibility. Polyacrylonitrile (PAN) provides excellent chemical resistance and a stable structure.

[0016] In one exemplary embodiment, the structure for extracorporeal blood perfusion may be one or more of porous microbeads, fibers, or membrane carriers for extracorporeal blood perfusion.

[0017] In one exemplary embodiment, the membrane carrier may be a dialysis membrane or an ultrafiltration membrane.

[0018] In one exemplary embodiment, the structure for in vitro blood perfusion can scavenge reactive oxygen species through the catalytic action of cerium dioxide nanoparticles.

[0019] In one exemplary embodiment, the cerium dioxide nanoparticles can be bound, attached to, or loaded onto the surface of the carrier through chemical bonding methods such as electrostatic bonding and covalent bonding, or through physical adsorption methods after surface modification.

[0020] In one exemplary embodiment, the structure for in vitro blood perfusion may be coated with one or more materials selected from the group consisting of PEG (polyethylene glycol) and PVP (polyvinylpyrrolidone).

[0021] On the other hand, the present invention provides an extracorporeal blood perfusion tube including the aforementioned structure for extracorporeal blood perfusion.

[0022] In another aspect, the present invention provides an extracorporeal blood purification device including the aforementioned extracorporeal blood perfusion tube.

[0023] In another aspect, the present invention provides an in vitro blood purification method comprising the following steps.

[0024] (a) The blood separated from the subject is brought into contact with the structure for in vitro blood perfusion; (b) Using the structure to remove reactive oxygen species from the blood; and (c) Recover the blood that has been cleared of the reactive oxygen species.

[0025] In another aspect, the present invention provides a method for preparing a structure for in vitro blood perfusion, comprising the following steps.

[0026] (a) Preparation of a support capable of loading cerium dioxide nanoparticles; and (b) Incorporating cerium dioxide nanoparticles into the structure.

[0027] In one exemplary embodiment, step (a) may include a step of modifying the surface of the prepared carrier.

[0028] In one exemplary embodiment, the method may further include the step of coating the surface of a structure for in vitro blood perfusion with cerium dioxide nanoparticles to enhance blood compatibility.

[0029] The effects of the invention On one hand, the present invention can effectively remove reactive oxygen species (ROS) that cause inflammation and damage from the blood in vitro by attaching cerium dioxide nanoparticles to a carrier capable of loading cerium dioxide nanoparticles, preferably a polymer-based carrier, and more preferably a carrier of various polymer-based carriers (such as, preferably, porous microbeads, fibers, or membrane carriers) that can be functionalized to load cerium dioxide nanoparticles. Furthermore, the use of a polymer-based carrier can effectively load cerium dioxide nanoparticles and prevent cerium dioxide nanoparticles or fragments of structures containing them from detaching from the structure used for in vitro blood perfusion and flowing into the bloodstream.

[0030] Furthermore, the extracorporeal blood purification device, constructed by circulating the patient's blood through an extracorporeal blood perfusion tube including such a structure for extracorporeal blood perfusion, can rapidly and extensively remove reactive oxygen species under both aqueous and real blood conditions. Therefore, this invention can be effectively applied to advanced extracorporeal blood perfusion therapy systems.

[0031] On the other hand, the present invention achieves continuous reactive oxygen species (ROS) scavenging. In particular, to address the problem of difficulty in continuously scavenging ROS under in vitro perfusion conditions, the present invention achieves continuous ROS scavenging capability by using a catalyst (cerium dioxide nanoparticles) instead of an adsorbent.

[0032] On the other hand, it is difficult to ensure the safety of inorganic nanoparticles such as cerium dioxide nanoparticles when they are infused into the body, so it is difficult to develop them into drugs. However, the present invention involves perfusing blood outside the body to remove reactive oxygen species, so its advantage lies in ensuring safety.

[0033] On another front, the present invention provides the following effects: It maximizes the catalytic properties of cerium dioxide nanoparticles to rapidly and effectively neutralize excess reactive oxygen species in the blood, thereby aiding in the treatment of sepsis and various inflammatory diseases. Furthermore, compared to rigid materials such as silica, the use of polymer-based soft carriers not only allows for more effective loading of cerium dioxide nanoparticles but also helps prevent the separation or elution of cerium dioxide nanoparticles or fragments of structures containing them from the structure used for in vitro hemoperfusion and their subsequent flow into the bloodstream. Moreover, while using polymer carriers, excellent mechanical properties and durability are maintained, ensuring stable performance during prolonged hemoperfusion.

[0034] On the other hand, the experimental results using the structure of the present invention for in vitro blood perfusion not only proved its efficacy in scavenging reactive oxygen species (ROS), but also demonstrated its stability and therapeutic effect in a real blood environment, which has the advantage of high feasibility for clinical application in sepsis and other inflammatory diseases.

[0035] On the other hand, unlike traditional extracorporeal perfusion therapy that nonspecifically adsorbs all substances that may be beneficial to the patient (such as anti-inflammatory cytokines), the present invention selectively removes reactive oxygen species, which are the root cause of inflammatory-damage responses, thereby solving the problems of traditional extracorporeal blood perfusion devices.

[0036] On the other hand, the present invention can remove increased reactive oxygen species in severe diseases associated with systemic inflammatory response syndrome, including sepsis, through extracorporeal perfusion, improve severity indicators (such as blood pressure and vasopressor dosage), and increase survival rates.

[0037] On the other hand, the structure of the present invention for extracorporeal blood perfusion can be easily integrated into conventional blood perfusion devices and tube systems, facilitating application in various therapeutic environments.

[0038] On the other hand, the structure of the present invention for in vitro blood perfusion can minimize non-specific protein adsorption and thrombus formation when in contact with blood by coating the surface with substances such as PVP, and inhibit biological reactivity. Attached Figure Description

[0039] Figures 1 to 3 The results show the loading conditions of PS / DVB microbeads-CeNPs according to an embodiment of the present invention. Figure 1 It shows the chemical formula introduced by sulfonation. Figure 2 This is a photograph showing the sulfonation treatment of PS / DVB microbeads. Figure 3 This is a characteristic peak diagram showing the introduction of sulfonyl (-SO3H) groups in sulfonated PS / DVB microbeads.

[0040] Figures 4 to 6 These are optical microscope and scanning electron microscope (SEM) images of the microbeads according to embodiments of the present invention. Figure 4 It is PS / DVB microbeads. Figure 5 It is sulfonated PS / DVB microbeads (S-PS / DVB). Figure 6 It is a sulfonated PS / DVB microbead loaded with cerium dioxide (S-PS / DVB@Ce).

[0041] Figure 7 This is an EDS elemental mapping image from an embodiment of the present invention, showing the results of EDS (Energy Dispersive X-ray Spectroscopy) analysis of S-PS / DVB@Ce microspheres.

[0042] Figure 8 The images shown are of S-PS / DVB microbeads with sulfonated groups (pre-sulfonated) and rich in mesoporous and macroporous structures, as observed by scanning electron microscopy (SEM) in this embodiment of the invention.

[0043] Figure 9 Is with Figure 8 The EDS element mapping results of microbeads that are identical to those used.

[0044] Figure 10 The images shown are scanning electron microscope (SEM) images and energy dispersive X-ray spectroscopy (EDS) analysis results of the structure (S-PS fiber@Ce) in which sulfonated polystyrene fiber (S-PS fiber) is loaded with cerium dioxide nanoparticles in an embodiment of the present invention.

[0045] Figure 11 These are images taken with an optical microscope showing the dimensional changes of polyethersulfone (PES)-based microspheres prepared according to embodiments of the present invention under different needle specifications.

[0046] Figure 12 This is an optical microscope image showing the size variation of PES / MCF composite microspheres prepared by mixing mesocellular silica foam (MCF) with PES under different needle specifications in an embodiment of the present invention.

[0047] Figure 13 This is a quantitative comparison of the average diameter of PES and PES / MCF microspheres prepared using a 20G needle in the embodiments of the present invention.

[0048] Figure 14 This is a schematic diagram illustrating the loading status and characteristic confirmation of cerium dioxide nanoparticles in PES-based microspheres according to an embodiment of the present invention.

[0049] Figure 15 These are optical microscope images and scanning electron microscope (SEM) images of PES microbeads prepared according to embodiments of the present invention.

[0050] Figure 16 These are optical microscope and SEM images of PES@Ce microspheres directly loaded with cerium dioxide nanoparticles in an embodiment of the present invention.

[0051] Figure 17 These are optical microscope and SEM images of PES / MCF microbeads containing MCF prepared in the embodiments of the present invention.

[0052] Figure 18 The images shown are optical microscope and SEM images of (PES / MCF)@Ce microspheres prepared by loading cerium dioxide nanoparticles onto composite microspheres including MCF in this embodiment of the invention.

[0053] Figure 19 This is the result of UV-Vis absorbance and quantitative analysis in the embodiments of the present invention, confirming that cerium dioxide nanoparticles (CeNPs) have been loaded onto PES and PES / MCF microspheres.

[0054] Figure 20 This is the result of observing the surface of the PES membrane using a scanning electron microscope (SEM) in an embodiment of the present invention.

[0055] Figure 21 This is the EDS mapping analysis result of PES-membrane@Ce loaded with cerium dioxide nanoparticles in the PES film in the embodiment of the present invention.

[0056] Figure 22 This is a photograph of the process of electrospinning polyacrylonitrile (PAN) dissolved in DMF in an ethanol / water mixed solvent, as described in an embodiment of the present invention.

[0057] Figure 23 These are scanning electron microscope (SEM) images of polyacrylonitrile (PAN) microbeads in embodiments of the present invention.

[0058] Figure 24 The surface modification of PAN-based microspheres in an embodiment of the present invention is shown.

[0059] Figure 25 This is a vibration peak diagram that confirms surface modification in the embodiments of the present invention.

[0060] Figure 26 The EDS mapping results of PAN-COOH microspheres loaded with cerium dioxide nanoparticles in an embodiment of the present invention are shown.

[0061] Figure 27 The results compare the reactive oxygen species (ROS) scavenging performance of PS / DVB-based microbeads in the embodiments of the present invention.

[0062] Figure 28 This is an evaluation result of the ROS removal capacity of porous polystyrene fiber (PS fiber) and sulfonated polystyrene fiber loaded with cerium dioxide nanoparticles (S-PS@Ce fiber) in the embodiments of the present invention.

[0063] Figure 29 The evaluation results of the ROS scavenging efficacy of PES-based microspheres loaded with cerium dioxide nanoparticles (CeNPs) in the embodiments of the present invention are shown.

[0064] Figure 30 This illustrates the results of ROS scavenging persistence when using CeNP-loaded microbead substrates to repeatedly perfuse 0.2 mM hydrogen peroxide (H2O2) solution in an embodiment of the present invention.

[0065] Figure 31 The results show a comparison of the ROS removal efficiency of a PES membrane and a PES membrane loaded with cerium dioxide nanoparticles (CeNPs) (PES@Ce) in accordance with embodiments of the present invention.

[0066] Figure 32 This figure shows the evaluation results of hydrogen peroxide (H2O2) scavenging performance based on the surface modification of PAN-based microspheres and the loading of cerium dioxide nanoparticles (CeNPs) in the embodiments of the present invention.

[0067] Figure 33 This demonstrates the results showing that the blood perfusion system of this invention significantly improved survival rates in an animal model of severe sepsis.

[0068] Figure 34 This demonstrates that the hemoperfusion system of this invention significantly improved hypotension in an animal model of severe sepsis. Detailed Implementation

[0069] The exemplary embodiments of the present invention will now be described in detail.

[0070] In this specification, "porous" refers to a structure that includes mesopores (2 nm to 50 nm) and macropores (greater than 50 nm) to effectively load cerium dioxide nanoparticles. These pores increase the internal or external surface area of ​​the carrier, expanding the contact area with the nanoparticles. In other words, porous structures can provide a larger surface area, allowing cerium dioxide nanoparticles to come into contact with as much reactive oxygen species (ROS) as possible.

[0071] In an exemplary embodiment of the present invention, cerium dioxide nanoparticles are used in a structure for extracorporeal blood perfusion. The reason for using cerium dioxide nanoparticles in the structure for extracorporeal blood perfusion is their unique redox cycling capability. Cerium (Ce) can be in an oxidation state of +3 (Ce... 3+ ) and +4 (Ce 4+ Cerium dioxide nanoparticles can freely convert between various reactive oxygen species (ROS), such as superoxide, hydrogen peroxide, hydroxyl radicals, and hypochlorite. In particular, cerium dioxide nanoparticles exhibit high reactivity due to the oxygen vacancies on their surface, making them a self-catalytic antioxidant capable of repeatedly scavenging ROS. Furthermore, unlike traditional antioxidants, they are not consumed and maintain a stable long-term effect.

[0072] Therefore, an exemplary embodiment of the present invention relates, from one perspective, to a structure for in vitro blood perfusion, comprising cerium dioxide nanoparticles and a carrier loading the cerium dioxide nanoparticles.

[0073] In one exemplary embodiment, the carrier may be porous microspheres.

[0074] In one exemplary embodiment, the carrier may be a porous fiber.

[0075] In one exemplary embodiment, the carrier may be a membrane carrier. The membrane carrier may be a semi-permeable polymer membrane (such as a dialysis membrane, a high-flux membrane, or an ultrafiltration membrane), for example, with an average pore size of approximately 5 nm to 50 nm or a molecular weight cutoff (MWCO) of approximately 10,000 Da to 100,000 Da. Furthermore, in one exemplary embodiment, the membrane carrier may include a hollow fiber membrane or a flat sheet membrane carrier for conventional hemodialysis, high-flux hemodialysis, or continuous renal replacement therapy (CRRT).

[0076] In one exemplary embodiment, the carrier preferably comprises a polymer. Specifically, the carrier preferably comprises a polymer, and includes more than 50 wt% polymer (which can be defined as a polymer-based material). Compared to rigid materials such as silica, using a soft carrier comprising a polymer, or preferably a polymer-based material, not only allows for more effective loading of cerium dioxide nanoparticles, but also helps prevent cerium dioxide nanoparticles or fragments of structures containing them from separating from or eluting from the structure used for in vitro blood perfusion and flowing into the bloodstream.

[0077] In one exemplary embodiment, the carrier may be composed of 50 wt% or more, 60 wt% or more, 70 wt% or more, 80 wt% or more, 90 wt% or more of a polymer, or may be composed of polymers alone.

[0078] In one exemplary embodiment, the carrier is polymer-based (i.e., the polymer is 50 wt% or more) and may additionally contain mesocellular silica foam (MCF).

[0079] In one exemplary embodiment, the polymer may be a polymer that can be functionalized to achieve loading of cerium dioxide nanoparticles.

[0080] For example, the polymer may be chitosan, chitin, polyethylene, polyacrylonitrile, polyvinylidene fluoride, polysulfone, polyethersulfone, polystyrene, polyvinyl alcohol, polymethyl methacrylate, cellulose, or cellulose acetate, etc.

[0081] The chitosan is a natural polymer with excellent biocompatibility and antibacterial properties, which can enhance ROS scavenging effects. The chitin is a precursor to chitosan, providing biocompatibility and biodegradability, making it suitable for various biomedical applications.

[0082] The polyethylene (PE) has excellent chemical stability and durability, and has the advantage of enabling low-cost large-scale production.

[0083] The polyacrylonitrile (PAN) possesses excellent mechanical strength and chemical resistance, providing a stable structure.

[0084] The polyvinylidene fluoride (PVDF) provides high chemical stability and heat resistance, and has the advantage of being able to be used stably even under extreme conditions.

[0085] The polysulfone (PSU) has high strength and heat resistance, and provides excellent chemical stability.

[0086] The polyethersulfone (PES) has similar properties to PSU and exhibits excellent biocompatibility.

[0087] The polystyrene (PS) can be incorporating various functional groups, which has the advantage of enhancing adsorption capacity.

[0088] The polyvinyl alcohol (PVA) provides excellent biocompatibility and water solubility, and has the advantage of being able to be processed into various forms.

[0089] The polymethyl methacrylate (PMMA) provides high transparency and biocompatibility, making it suitable for medical devices.

[0090] Cellulose is a natural material that offers biocompatibility and environmental friendliness. Furthermore, cellulose acetate, in addition to its biocompatibility, is also cost-effective.

[0091] The above-mentioned polymer materials can be used for porous microspheres, porous fibers, and membrane carriers. In particular, when used as membrane carriers, the cellulose acetate, polysulfone (PSU), and polyethersulfone (PES) are preferably used.

[0092] In one exemplary embodiment, the carrier has a mesoporous or macroporous structure, which can provide a large surface area to introduce a large number of cerium dioxide nanoparticles.

[0093] In one exemplary embodiment, the carrier can be functionalized by introducing functional groups such as sulfone groups that can electrostatically bond with cerium dioxide nanoparticles to induce strong electrostatic bonding with the cerium dioxide nanoparticles, or by introducing functional groups such as amine groups or carboxyl groups that can form covalent bonds to form strong covalent bonds with the cerium dioxide nanoparticles. This physical structure and chemical functionalization allows cerium dioxide nanoparticles to be stably and massively loaded onto the carrier, effectively preventing the separation or elution of cerium dioxide nanoparticles or fragments of structures containing them from the blood perfusion environment.

[0094] In one exemplary embodiment, the polymer may include one or more polymers selected from the group consisting of polystyrene (PS), polyethersulfone (PES), and polyacrylonitrile (PAN), which possess excellent mechanical strength and biocompatibility in blood perfusion environments, and have the property of being functionalized to achieve loading of cerium dioxide nanoparticles. Specifically, as previously mentioned, polystyrene (PS) readily incorporates various functional groups, particularly beneficial for loading and surface modification of CeNPs, while polyethersulfone (PES) is characterized by its inherent hydrophilicity and excellent biocompatibility. Polyacrylonitrile (PAN) provides excellent chemical resistance and a stable structure.

[0095] In one exemplary embodiment, the structure for in vitro blood perfusion can scavenge reactive oxygen species (ROS) through the catalytic action of cerium dioxide nanoparticles. In other words, the cerium dioxide nanoparticles exhibit excellent antioxidant properties, capable of continuously neutralizing ROS through autocatalysis.

[0096] In one exemplary embodiment, the cerium dioxide nanoparticles can be bound, attached to, or loaded onto the surface of the carrier through chemical bonding methods such as electrostatic bonding and covalent bonding, or through physical adsorption methods after surface modification.

[0097] In one exemplary embodiment, the structure for in vitro blood perfusion may have the following features: uniformly dispersing cerium dioxide nanoparticles throughout the carrier to maximize the removal efficiency of reactive oxygen species.

[0098] In one exemplary embodiment, the size (diameter) of the porous microspheres can be 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 less than 1500 μm, less than 1400 μm, less than 1300 μm, less than 1200 μm, less than 1100 μm, less than 1000 μm, less than 900 μm, less than 800 μm, less than 700 μm, less than 650 μm, less than 600 μm, less than 550 μm, but is not limited thereto. For example, the size (diameter) of the porous microspheres can be 1 μm to 1500 μm, or 10 μm to 1000 μm, or 100 μm to 1000 μm. The size (diameter) of the porous microbeads can be adjusted according to the cylinder size and effective blood perfusion. In a non-limiting example, the size (diameter) of the porous microbeads can be set to be larger than blood cells, such as greater than 10 μm.

[0099] In one exemplary embodiment, the reactive oxygen species may be one or more selected from the group consisting of superoxide anion, hydrogen peroxide, and hydroxyl radical, but is not limited thereto.

[0100] In one exemplary embodiment, the pore volume of the porous microspheres may be 0.1 cm³. 3 / g to 13.5cm 3 / g, preferably 0.1cm 3 / g to 5.0cm 3 / g, more preferably 0.5cm 3 / g to 1.5cm 3 / g, but not limited thereto. Porous microspheres with the aforementioned pore volume can effectively load cerium dioxide nanoparticles and scavenge reactive oxygen species.

[0101] In one exemplary embodiment, the surface area of ​​the porous microspheres may be 100 μm. 2 / g to 1000m 2 / g, preferably 200m 2 / g to 900m 2 / g, more preferably 300m 2 / g to 800m 2 / g, but not limited thereto. Porous microspheres with the aforementioned surface area can effectively load cerium dioxide nanoparticles and scavenge reactive oxygen species.

[0102] In one exemplary embodiment, the porous fiber may be a polymer-based porous fiber or a porous fiber composed of polymers, with a diameter of approximately 10 μm to 50 μm. For example, it may be formed from polystyrene (PS), polypropylene (PP), polycaprolactam (PA-6), polyacrylonitrile (PAN), or copolymers thereof, and the surface may be chemically treated to introduce highly active functional groups or load nanoparticles. The fiber may have a large surface area and porosity, such as a porosity of less than 10%, such as approximately 0.1% to 10%, to ensure durability and flow properties.

[0103] In one exemplary embodiment, the membrane carrier may use a non-porous structure, but is preferably a porous structure. For example, it may be provided in the form of a hollow fiber membrane or a flat sheet membrane, or a semi-permeable polymer membrane, with the purpose of removing specific molecules from blood or plasma. The membrane is generally formed from polysulfone (PS), polyethersulfone (PES), polyvinylidene fluoride (PVDF), polymethyl methacrylate (PMMA), polyacrylonitrile (PAN), cellulose derivatives, etc. The physical properties of the membrane are, for example, an outer diameter of approximately 200 μm to 350 μm, an inner diameter of approximately 150 μm to 250 μm, a wall thickness of approximately 30 μm to 100 μm, a pore size of approximately 5 nm to 50 nm, and a porosity of approximately 30% to 80%. Furthermore, the molecular weight cutoff (MWCO) is approximately 10,000 Da to 100,000 Da, and the ultrafiltration coefficient (UF coefficient) is approximately 5 mL / h / mmHg / m 2 ~80mL / h / mmHg / m 2 The surface area is 0.2m². 2 ~2.5m 2 The membrane can be adjusted according to the therapeutic purpose. Furthermore, the membrane can be surface modified, for example, by coating it with a hydrophilic polymer (such as PVP), introducing heparin, or introducing functional groups for loading nanoparticles, to improve biocompatibility.

[0104] In one exemplary embodiment, the structure for in vitro blood perfusion may contain cerium dioxide nanoparticles at a concentration of 0.5 mg / g or higher. Here, "mg / g" refers to the amount (milligrams) of cerium dioxide nanoparticles per gram of carrier.

[0105] Specifically, the structure for extracorporeal blood perfusion may contain cerium dioxide nanoparticles at concentrations of 0.5 mg / g or higher, 1 mg / g or higher, 2 mg / g or higher, 3 mg / g or higher, 4 mg / g or higher, 5 mg / g or higher, 10 mg / g or higher, 15 mg / g or higher, 20 mg / g or higher, 30 mg / g or higher, 40 mg / g or higher, or 50 mg / g or lower, but is not limited thereto. For example, in a container containing 100 mg / g or 200 mg / g of cerium dioxide nanoparticles, the structure for extracorporeal blood perfusion can effectively perform extracorporeal blood perfusion and reactive oxygen species scavenging. In one exemplary embodiment, the structure for extracorporeal blood perfusion may be coated with one or more materials selected from the group consisting of PEG (polyethylene glycol) and PVP (polyvinylpyrrolidone). The coating can prevent the structure used for in vitro hemoperfusion from reacting with blood, thereby inhibiting hemolysis or coagulation and improving the blood compatibility of the structure. In this regard, structures for in vitro hemoperfusion coated with PEG (polyethylene glycol) are preferred, and structures for in vitro hemoperfusion coated with PVP (polyvinylpyrrolidone) are further preferred.

[0106] From another perspective, the present invention relates to an extracorporeal blood perfusion tube comprising the aforementioned structure for extracorporeal blood perfusion.

[0107] In one exemplary embodiment, the volume fraction of the structure for extracorporeal blood perfusion relative to the entire extracorporeal blood perfusion cylinder can be 50% or more, 55% or more, 60% or more, 65% or more, or 70% or less, or less than 90%, 85% or less, or less than 80%. The volume fraction of the structure for extracorporeal blood perfusion relative to the entire extracorporeal blood perfusion cylinder can be from 50% to 90%, but is not limited thereto. Extracorporeal blood perfusion and reactive oxygen species removal can be effectively performed at said volume fraction.

[0108] From another perspective, the present invention relates to an extracorporeal blood purification device including the aforementioned extracorporeal blood perfusion tube.

[0109] From another perspective, this invention relates to an in vitro blood purification method comprising the following steps: (a) The blood separated from the subject is brought into contact with the structure for in vitro blood perfusion; (b) Using the aforementioned structure for extracorporeal blood perfusion to remove reactive oxygen species from the blood; and (c) Recover the blood that has been cleared of the reactive oxygen species.

[0110] In one exemplary embodiment, the removal of the reactive oxygen species can be carried out for 2 to 24 hours, but is not limited to this, and the structure for in vitro blood perfusion achieves continuous reactive oxygen species removal by using a catalyst (cerium dioxide nanoparticles) instead of an adsorbent. Therefore, reactive oxygen species can be continuously removed for the duration required by the subject without time constraints.

[0111] In another aspect, the present invention relates to a method for preparing a structure for in vitro blood perfusion, comprising the following steps.

[0112] (a) Preparing a carrier comprising one or more polymers selected from the group consisting of polystyrene (PS), polyethersulfone (PES), and polyacrylonitrile (PAN); and (b) Incorporating cerium dioxide nanoparticles into the carrier.

[0113] In one exemplary embodiment, step (a) may include a step of modifying the surface of the prepared carrier.

[0114] In one exemplary embodiment, step (a) may modify the carrier surface using an formulation comprising one or more functional groups selected from the group consisting of amine, sulfonic acid, thiol, carboxylic, hydroxyl, or epoxy groups.

[0115] In one exemplary embodiment, the method may further include the following steps: (c) coating a polymer onto the surface of a carrier to which cerium dioxide nanoparticles are attached.

[0116] In one exemplary embodiment, in step (c), one or more of the group consisting of PEG (polyethylene glycol) and PVP (polyvinylpyrrolidone) may be coated, but are not limited thereto.

[0117] In one exemplary embodiment, the method may further include a coating step with heparin and / or heparin derivatives, but is not limited thereto.

[0118] The invention will now be described in more detail by way of examples. These examples are for illustrative purposes only, and it will be apparent to those skilled in the art that the scope of the invention should not be construed as limited to these examples.

[0119]

Example

[0120] (1-2) Preparation, functionalization and loading of CeNPs on carrier materials Loading of directly sulfonated polystyrene (PS) microspheres and cerium dioxide nanoparticles Macroporous polystyrene-divinylbenzene (PS-DVB) based porous beads are used as the substrate. These beads have sufficient internal or external surface area to effectively load CeNPs and have the property of allowing the introduction of functional groups or modification of the surface.

[0121] Specifically, the representative physicochemical properties of the beads are as follows: The diameter ranges from 300 μm to 800 μm, the pore volume is 0.3 mL / g, and the surface area is 950 m². 2 The average pore diameter of the mesopores / macropores / transfer pores is 220 Å, and the average pore diameter of the micropores is 15 Å. The microbeads are immersed in a concentrated sulfuric acid (approximately 95%–98%) solution and sulfonated at 80°C for 3 hours. Subsequently, residual acid is removed by thorough washing with distilled water, and the microbeads are dried.

[0122] Subsequently, cerium dioxide nanoparticles (CeNPs) were loaded as follows.

[0123] One gram of sulfonated PS-DVB beads was mixed with 40 mL of CeNP aqueous solution with a concentration of 5 mg / mL, and stirred at room temperature for 24 hours to load cerium dioxide nanoparticles. After loading, the mixture was washed five times with deionized water to remove unadsorbed particles, dispersed in deionized water, and stored under cold conditions.

[0124] Loading of presulfonated polystyrene (PS) microspheres and cerium dioxide nanoparticles In another embodiment, porous beads based on PS-DVB with introduced sulfonic acid functional groups were used. These beads possess strong acidic cation exchange properties and can load cerium dioxide nanoparticles without separate surface treatment.

[0125] Specifically, the representative physicochemical properties of the beads are as follows: diameter 300μm~1200μm, pore volume 0.3mL / g, and surface area 660m². 2 / g, the average pore diameter of mesopores / macropores / transmission pores is 650 Å, and the average pore diameter of micropores is 15 Å.

[0126] The loading process of cerium dioxide nanoparticles was carried out under the same conditions as described above (5 mg / mL CeNP aqueous solution, stirring for 24 hours, washing 5 times, and then refrigerating).

[0127] Loading of polystyrene (PS) fibers and cerium dioxide nanoparticles Commercially available porous polystyrene fibers or polystyrene-divinylbenzene (PS / DVB) fibers are used as the substrate. The fibers have sufficient internal or external surface area to effectively load CeNPs and possess properties that allow for the introduction of functional groups or modification of the surface.

[0128] Specifically, the porous fibers may have a pore volume of 0.3 mL / g or higher and a pore size of 600 m³ / g. 2 The fibers have a surface area of ​​over / g and an average pore diameter of over 500 Å. The fibers undergo a sulfonation process to introduce negatively charged sulfonyl groups onto the surface. The fibers are immersed in a concentrated sulfuric acid (approximately 95%–98%) solution for sulfonation at 80°C for 3 hours. Subsequently, residual acid is removed through a thorough deionized water washing process, and the fibers are dried.

[0129] Subsequently, cerium dioxide nanoparticles (CeNP) were loaded as follows.

[0130] Sulfonated polystyrene fibers were cut into 100mm × 100mm pieces, and then mixed with 40mL of cerium dioxide nanoparticles at a concentration of 5mg / mL (per fiber). The mixture was stirred at room temperature for 24 hours to load the cerium dioxide nanoparticles. After loading, the fibers were washed five times with deionized water (30 minutes each time) to remove unadsorbed cerium dioxide nanoparticles, and then dried with ethanol under vacuum. Subsequently, the sulfonated polystyrene fibers loaded with cerium dioxide nanoparticles were cryopreserved.

[0131] Loading of polyethersulfone (PES) based microspheres and cerium dioxide nanoparticles (1) Loading of PES microspheres and CeNP PES microspheres were prepared by electrospinning a 10 wt% PES solution (solvent: DMF) in a 1:1 ethanol / water mixture. (The electrospinning voltage was 7 kV, and the PES solution flow rate was 0.5 mL / min.) The prepared PES beads (10 mL) were mixed with 40 mL of cerium dioxide nanoparticle (CeNP) aqueous solution with a concentration of 5 mg / mL and stirred at room temperature for 24 hours to load CeNP.

[0132] Subsequently, it was washed five times with deionized water, dispersed in deionized water, and stored in the refrigerator for later use.

[0133] (2) Loading of PES / MCF composite microspheres and CeNP To facilitate the loading of cerium dioxide nanoparticles, 1 g of MCF (mesocellular silica foam) was dispersed in a 10 wt% PES solution (DMF, 20 mL), followed by electrospinning in a 1:1 ethanol / water mixture to prepare PES / MCF composite microspheres. (Electrospinning voltage: 7 kV; PES solution flow rate: 0.5 mL / min) The prepared PES / MCF beads (10 mL) were mixed with CeNP aqueous solution (5 mg / mL, 40 mL) under the same conditions as above, loaded for 24 hours, washed 5 times, and then dispersed in deionized water and stored under cold.

[0134] Polyethersulfone (PES) dialysis membrane and cerium dioxide nanoparticle loading In this embodiment, a polyethersulfone (PES) flat sheet membrane carrier is used as the semi-permeable polymer membrane. The membrane has the following specifications: a diameter of 25 mm, a pore size of approximately 30 nm, and a thickness of 110 μm. It was purchased and used from commercially available flat-sheet SEM (Surface Engineered Membrane) membrane products.

[0135] This membrane is a structure that is substantially similar to a conventional hemodialysis membrane in terms of its morphology, pore size and the materials used. It is used as an exemplary model for implementing a dialysis membrane to evaluate the feasibility of loading cerium dioxide nanoparticles according to the present invention.

[0136] 40 mL of cerium dioxide nanoparticles with a concentration of 5 mg / mL were mixed and stirred at room temperature for 24 hours to load the cerium dioxide nanoparticles. After loading, the nanoparticles were washed five times with deionized water (30 minutes each time) to remove unadsorbed cerium dioxide nanoparticles, and then the water was removed with ethanol and dried under vacuum.

[0137] Loading of polyacrylonitrile (PAN) microspheres and cerium dioxide nanoparticles Porous microspheres were directly prepared using PAN polymers (e.g., an average molecular weight M of approximately 150,000). PAN microspheres were prepared by electrospinning a 3 wt% PAN solution (solvent: DMF) in a 1:1 ethanol / water mixture (electrospinning voltage: 7 kV, PAN solution flow rate: 0.5 mL / min). To modify the surface carboxyl groups, the prepared PAN beads (7.5 mL) were dehydrated and then mixed with 40 mL of 1 M NaOH, stirred for 24 hours. Subsequently, they were washed five times with deionized water (30 minutes each time). The PAN beads with negatively charged carboxyl groups turned orange.

[0138] To load cerium dioxide nanoparticles onto carboxyl-modified PAN beads, the prepared beads (10 mL) were mixed with 5 mg / mL cerium dioxide nanoparticles (40 mL), loaded for 24 hours, washed 5 times, and then dispersed in deionized water and stored under cold conditions.

[0139] (1-3) Coating process (PVP) PVP (polyvinylpyrrolidone) is used to coat microbeads, fibers, and membranes loaded with CeNPs to improve blood compatibility and prevent CeNPs from detaching.

[0140] PVP coating Microbeads, fibers, or membranes loaded with CeNPs (e.g., 1 g) were mixed with a 5 mg / mL PVP solution (20 mL) for 6 hours, washed with deionized water, and dried in a vacuum oven. The resulting P-Ce-polymer carrier (microbeads, fibers, or membranes) was then refrigerated for later use.

[0141] (1-4) Preparation and system composition of the blood perfusion tube The outer casing consists of an upper section, a barrel section, and a lower section. The various parts of the outer casing were designed using Fusion 360 software (Autodesk, CA, USA) and fabricated using an SLA-based ProJet 7000 3D printer (3D Systems, SC, USA). Accura ClearVue was used as the resin to visualize the interior of the casing. O-rings were inserted at the joints between the various parts of the outer casing to prevent leakage. 200 mg of blood perfusion microbeads were filled into the assembled outer casing to assemble the blood perfusion cylinder.

[0142] (1-5) Animal experiments with sepsis The experimental groups consisted of a P-Ce-PS / DVB (cerium dioxide-loaded and PVP-coated polystyrene-divinylbenzene microbeads) treatment group and an untreated group receiving only standard treatment. Male Sprague-Dawley rats aged 9–14 weeks and weighing 300–450 g were used. For anesthesia induction, sedation was initially achieved with 4% isoflurane, followed by intramuscular injections of tiletamine / zolazepam (30 mg / kg) and xylazine (10 mg / kg). Subsequently, endotracheal intubation was performed using a 16G catheter, and mechanical ventilation was initiated using an Inspira Advanced Safety Ventilator. Sedation was maintained by 0.5%–1% isoflurane connected to the ventilation circuit, and tramadol (500 mcg) was administered subcutaneously for analgesia.

[0143] Under aseptic conditions, cannulas were inserted into the left common carotid artery, right common femoral artery, and left common femoral vein using 24G catheters. Blood pressure was monitored throughout the experiment using pressure sensors and monitors connected to the arterial catheters. Body temperature was monitored using a rectal probe and regulated and maintained at 36.5°C–37.5°C using an infrared heater. All fluids and medications were administered via a three-way stopcock valve connected to the left femoral vein catheter. The hemoperfusion circuit began with the left common carotid artery cannula, connected via a catheter to the hemoperfusion cartridge, then via an additional catheter to the three-way stopcock valve for infusion and sampling, and finally connected to the left common femoral vein catheter. The circuit was initially filled with 100 mL of heparinized saline (50 IU / mL), followed by flushing 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 a 10-minute infusion of 30 mL / kg of saline for fluid resuscitation. Hemoperfusion 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. After perfusion, the blood was returned to the left common femoral vein. When the mean arterial pressure (MAP) was below 60 mmHg, norepinephrine was infused at a flow rate of 0.1 mcg / kg / min, and the flow rate could be adjusted to a maximum of 2 mcg / kg / min based on the MAP.

[0144] The log-rank (Mantel-Cox) test was used to assess the differences in survival curves among the groups. A mixed effects model, which considers both fixed effects (treatment group) and randomized effects (inter-individual differences), was used to analyze the differences in blood pressure changes over time among the groups.

[0145] 2. Experimental Results (2-1) Characteristic analysis results of the prepared microspheres, fibers and membranes Figures 1 to 3 The results show the loading conditions of PS / DVB microbeads-CeNPs according to an embodiment of the present invention. Figure 1 It shows the chemical formula introduced by sulfonation. Figure 2 This is a photograph showing the sulfonation treatment of PS / DVB microbeads. Figure 3 This is a characteristic peak diagram showing the introduction of sulfonyl (-SO3H) groups in sulfonated PS / DVB microbeads.

[0146] As in Figure 3 As can be seen, FTIR spectroscopy analysis confirmed the successful display of characteristic peaks (S=O asymmetric / symmetric stretching vibrations, range 1000 cm⁻¹) indicating the introduction of sulfonyl (-SO₃H) groups in sulfonated PS / DVB microspheres. -1 ~1200cm -1 This confirms that the surface functionalization (sulfonation) of PS / DVB microspheres has been effectively achieved.

[0147] Figures 4 to 6 These are optical microscope and scanning electron microscope (SEM) images of the microbeads according to embodiments of the present invention. Figure 4 It is PS / DVB microbeads. Figure 5 It is sulfonated PS / DVB microbeads (S-PS / DVB). Figure 6 These are sulfonated PS / DVB microspheres loaded with cerium dioxide (S-PS / DVB@Ce). This indicates that after sulfonation treatment and loading with cerium dioxide nanoparticles, the microspheres still maintain a uniform spherical morphology, and the cerium dioxide nanoparticles are uniformly attached to the surface.

[0148] Figure 7 This is an EDS elemental mapping image from an embodiment of the present invention, showing the results of EDS (Energy Dispersive X-ray Spectroscopy) analysis of S-PS / DVB@Ce microspheres. (See image below.) Figure 7 As can be seen from the image, sulfur (S) elemental mapping (left) confirms the widespread distribution of sulfur on the sulfonated polymer surface. Cerium (Ce) elemental mapping (middle) clearly confirms that cerium dioxide nanoparticles (Ce) are effectively loaded and uniformly distributed on the microsphere surface. This strongly confirms that sulfonation has been successfully achieved, and that the positively charged CeNPs are stably attached to the introduced sulfonic acid functional groups via electrostatic attraction.

[0149] Figure 8 Images of S-PS / DVB microbeads that have been pre-sulfonated and rich in mesoporous and macroporous structures, as observed by scanning electron microscopy (SEM).

[0150] Surface (left image): While maintaining a smooth appearance, beads in the diameter range of 300μm to 1200μm showed subtle unevenness and porous patterns.

[0151] Cross-section (right figure): Confirms a thick, interwoven internal framework and a continuous mesoporous-macroporous network, reflecting bead properties with an average pore size increased to approximately 650 Å. This structure enhances the permeation and loading capacity of cerium dioxide nanoparticles (CeNPs).

[0152] Figure 9 Is with Figure 8 The EDS element mapping results of microbeads that are identical to those used.

[0153] As in Figure 9 As can be seen, the sulfur (S) signal is uniformly distributed throughout the fiber skeleton, indicating that the sulfonic acid group -SO3H is present throughout the bead.

[0154] Furthermore, a uniform cerium (Ce) signal was even generated inside the beads, confirming that the cerium dioxide nanoparticles were stably loaded throughout the entire surface and pores of the beads. The simultaneous distribution of sulfur and cerium strongly suggests that all nanoparticles were tightly immobilized due to the electrostatic interaction between the negatively charged sulfonyl groups and the positively charged CeNPs.

[0155] Figure 10 This image presents scanning electron microscopy (SEM) images and energy-dispersive X-ray spectroscopy (EDS) analysis results of a structure (S-PS fiber@Ce) containing cerium dioxide nanoparticles loaded within sulfonated polystyrene (S-PS) fiber. The SEM image confirms well-formed fiber structure, and the EDS mapping results confirm that cerium dioxide (Ce) is uniformly distributed throughout the fiber surface. Furthermore, the presence of sulfur (S) and the distribution of Ce support the introduction of sulfonyl groups and the loading of cerium dioxide, respectively.

[0156] Figure 11 Images taken using an optical microscope show the dimensional changes of polyethersulfone (PES)-based microspheres prepared according to embodiments of the present invention under different needle gauges. As the needle gauge is changed to 22G, 20G, and 18G, the average diameter of the microspheres gradually increases, reaching its maximum of approximately 600 μm to 800 μm when using an 18G needle. Under all conditions, the microspheres maintain uniform sphericity.

[0157] Figure 12This is an optical microscope image showing the size variation of PES / MCF composite microspheres prepared by mixing mesocellular silica foam (MCF) with PES under different needle sizes, as described in an embodiment of the present invention. Similar to PES single polymer beads, the size of the composite microspheres also tends to increase with the increase of needle size, while maintaining excellent shape stability and sphericity overall.

[0158] Figure 13 This is a quantitative comparison of the average diameter of PES and PES / MCF microspheres prepared using a 20G needle. The PES / MCF composite microspheres exhibited a significantly smaller average diameter than the single PES polymer beads (****, p < 0.0001), indicating that variations in the composition of the polymer composite may affect the physical properties of the microsphere formation process.

[0159] Figure 14 These are schematic diagrams illustrating the loading and characteristic confirmation of cerium dioxide nanoparticles in PES-based microspheres according to embodiments of the present invention. The first schematic diagram on the left shows basic PES microspheres prepared by electrospinning a 10 wt% PES solution (DMF) in a 50% ethanol / water mixture. The second schematic diagram shows a structure (PES@Ce) where PES microspheres are immersed in a CeNP aqueous solution to physically adsorb cerium dioxide nanoparticles onto the bead surface. The third schematic diagram shows a composite microsphere (PES / MCF) prepared by electrospinning a mixture of mesocellular silica foam (MCF) into a PES solution. The fourth schematic diagram shows a (PES / MCF)@Ce structure prepared by adsorbing CeNPs onto the composite microspheres. The MCF contains a porous silica structure, which enhances the CeNP loading capacity.

[0160] Figure 15 These are optical microscope images and scanning electron microscope (SEM) images of PES microspheres prepared according to embodiments of the present invention. As seen in the optical images, the microspheres prepared from the single polymer PES maintain a uniform and delicate spherical morphology, with an average diameter of approximately 600 μm. SEM analysis confirmed that their outer surface is smooth, and their inner cross-section has a spongy porous structure with a uniformly distributed nanoscale pore structure. These microspheres were used as a control group for subsequent loading experiments.

[0161] Figure 16These are optical microscope and SEM images of PES@Ce microspheres directly loaded with cerium dioxide nanoparticles in an embodiment of the present invention. The optical microscope images confirm that they maintain a spherical structure similar to that of individual PES beads, and SEM analysis reveals that the cerium dioxide nanoparticles are finely attached to their surface. It also shows that the microspheres maintain a nanoporous structure in their inner cross-section, with cerium dioxide uniformly dispersed within the matrix without aggregation.

[0162] Figure 17 These are optical microscope and SEM images of the PES / MCF microspheres containing MCF prepared in this embodiment of the invention. The optical microscope images show sphericity and size similar to those of single PES polymer beads, while SEM analysis confirms that the MCF introduced into the PES matrix has resulted in a denser and more complex porous structure. This allows for both an increase in bead surface area and structural reinforcement.

[0163] Figure 18 These are optical microscope and SEM images of (PES / MCF)@Ce microspheres prepared by loading cerium dioxide nanoparticles onto composite microspheres including MCF in this embodiment of the invention. The optical microscope images confirm its excellent morphological stability, and the SEM analysis confirms that the cerium dioxide particles are uniformly attached to the surface. In the internal cross-section, the cerium dioxide is effectively loaded into the MCF structure, thus maintaining a porous channel structure while the cerium dioxide exists in an integrated form within the network structure.

[0164] Figure 19 This embodiment of the invention presents the results of UV-Vis absorbance and quantitative analysis confirming that cerium dioxide nanoparticles (CeNPs) have been loaded onto PES and PES / MCF microspheres. In the left-hand image, a characteristic absorption peak (approximately 260 nm) of CeNPs was observed in both PES and PES / MCF microspheres, indicating that CeNPs have been successfully loaded. The quantitative analysis results in the right-hand image confirm that, under the same volume (1 mL) conditions, PES / MCF microspheres loaded more CeNPs than PES microspheres.

[0165] Figure 20 This is the result of observing the surface of the PES membrane using a scanning electron microscope (SEM) in an embodiment of the present invention. The minute irregularities and porous surface confirmed from the SEM images indicate the inherent physical properties of the membrane, which are interpreted as suitable substrate structures for surface modification and functionalization of cerium dioxide nanoparticles (CeNPs).

[0166] Figure 21This is the EDS mapping analysis result of PES-membrane@Ce with cerium dioxide nanoparticles loaded in the PES film in this embodiment of the invention. The analysis results show that the Ce signal is uniformly distributed throughout the film, which qualitatively indicates that the cerium dioxide nanoparticles are effectively loaded on the surface. These characteristics support the conclusion that the film of this embodiment can be used as a substrate for achieving reactive oxygen species (ROS) scavenging function.

[0167] Figure 22 This illustrates a scenario in which, during the electrospinning of polyacrylonitrile (PAN) dissolved in DMF in an ethanol / water mixed solvent according to an embodiment of the invention, the internal DMF is rapidly removed through solvent exchange with the external solvent, while the insoluble PAN precipitates out in bead form. This confirms the formation of white PAN beads.

[0168] Figure 23 These are scanning electron microscope (SEM) images of polyacrylonitrile (PAN) microspheres in an embodiment of the present invention, showing the surface and cross-sectional structures of the microspheres. In the surface SEM image on the left, the PAN microspheres maintain a uniform spherical morphology, and a tiny porous surface structure is observed in the high-magnification image. The cross-sectional image on the right confirms a non-uniformly distributed porous channel structure throughout the interior, indicating that pores also form within the microspheres.

[0169] Such surface and internal structures can serve as a basic structure to facilitate the removal of reactive oxygen species (ROS) during in vitro blood perfusion by providing high specific surface area and diffusion pathways to effectively load functional nanomaterials such as cerium dioxide nanoparticles (CeNPs).

[0170] Figure 24 The surface modification of PAN-based microspheres in an embodiment of the present invention is illustrated. PAN is a polymer with nitrile groups (-CN). To load cerium dioxide nanoparticles (CeNPs), surface modification is required to impart a negative charge. Therefore, PAN microspheres are treated in an alkaline aqueous solution to hydrolyze the nitrile groups, thereby converting them into carboxyl groups (-COOH), which increases the negative charge on the surface of the microspheres. The color of the treated microspheres changes from white to orange, as observed by the naked eye, indicating that the surface modification has been successfully performed.

[0171] Figure 25 It can be confirmed that traditional PAN, under the influence of nitrile functional groups, reaches 2243 cm⁻¹ -1 The appearance of a vibration peak at 3354 cm⁻¹ indicates that the surface-modified PAN-COOH exhibits absorption at 3354 cm⁻¹ due to the OH vibration of the carboxyl group. -1 A broad peak appeared at 1664 cm⁻¹, confirming the typical C=O of the carboxyl group at 1664 cm⁻¹. -1A vibrational peak appears at this location, along with negatively charged COO. - At 1405cm -1 1564cm -1 The presence of symmetrical and asymmetrical peaks confirms that the surface has been modified with carboxyl groups.

[0172] Figure 26 The EDS mapping results of PAN-COOH microspheres loaded with cerium dioxide nanoparticles in an embodiment of the present invention are shown. The signals of nitrogen (N) and cerium (Ce) elements are distributed throughout the surface of the microspheres, indicating that the cerium dioxide nanoparticles have been effectively loaded.

[0173] Figure 27 This study compares the reactive oxygen species (ROS) scavenging performance of PS / DVB-based microbeads in the embodiments of the present invention. The control group (without cerium dioxide loading) and PS / DVB microbeads showed almost no H2O2 scavenging effect, but the S-PS / DVB@Ce microbeads loaded with cerium dioxide significantly reduced the H2O2 concentration. In particular, excellent ROS scavenging ability was confirmed under both direct sulfonation and pre-sulfonation methods, indicating that CeNP was effectively functionalized under both conditions.

[0174] Figure 28 This is an evaluation of the ROS removal capabilities of porous polystyrene fiber (PS fiber) and sulfonated polystyrene fiber loaded with cerium dioxide nanoparticles (S-PS@Ce fiber) in the embodiments of the present invention. Polystyrene fiber without cerium dioxide nanoparticles showed almost no ROS removal efficiency, while sulfonated polystyrene fiber loaded with cerium dioxide was confirmed to have a removal efficiency of approximately 90%.

[0175] Figure 29 The evaluation results of the ROS scavenging efficacy of PES-based microspheres loaded with cerium dioxide nanoparticles (CeNPs) in embodiments of the present invention are shown. Figure 29 As can be seen, the control group (PES and PES / MCF) without cerium dioxide loading had almost no ROS removal effect, while the PES@Ce and (PES / MCF)@Ce microbeads loaded with cerium dioxide removed about 80% of hydrogen peroxide (H2O2), showing significant ROS removal ability.

[0176] Figure 30This invention illustrates the sustained ROS scavenging performance of a microbead-based cartridge loaded with CeNPs during repeated perfusion with 0.2 mM hydrogen peroxide (H2O2) solution. 10 mL of H2O2 solution was repeatedly perfused into a cartridge containing 1 mL of microbeads at a rate of 1 mL / min, and the residual H2O2 concentration was measured in each cycle (0, 1, 3, 5, 7). The results show that both PES@Ce and PES / MCF@Ce microbeads maintained stable H2O2 scavenging capabilities during repeated perfusion, with PES / MCF@Ce exhibiting a slightly lower overall residual H2O2 concentration and slightly better ROS scavenging efficiency. This result indicates that cerium dioxide nanoparticles possess catalytic activity and retain ROS scavenging activity even after repeated use.

[0177] Figure 31 The results show a comparison of the ROS removal efficiency of a PES membrane and a PES membrane loaded with cerium dioxide nanoparticles (CeNPs) (PES@Ce) in accordance with embodiments of the present invention.

[0178] PES membranes without cerium dioxide loading showed almost no ability to remove hydrogen peroxide (H2O2), while PES@Ce membranes removed approximately 95% of H2O2, demonstrating excellent ROS removal efficiency. This indicates that the porous structure of PES membranes can achieve effective loading of CeNPs, and cerium dioxide-loaded membranes can be effectively utilized as materials for ROS removal.

[0179] Figure 32 This figure shows the evaluation results of hydrogen peroxide (H2O2) scavenging performance based on the surface modification of PAN-based microspheres and the loading of cerium dioxide nanoparticles (CeNPs) in the embodiments of the present invention.

[0180] The experimental group consisted of four conditions, each categorized as follows.

[0181] (1) PAN: Unmodified or unloaded pure PAN beads, (2) PAN@Ce: PAN beads directly loaded with CeNP. (3) PAN-COOH: PAN beads with carboxyl (-COOH) modified surface. (4) (PAN-COOH)@Ce: (PAN beads modified with carboxyl groups and loaded with CeNP).

[0182] Figure 32 The results show that loading CeNPs helps to remove H2O2, especially after modifying the PAN surface with carboxyl groups, when CeNPs are loaded, the H2O2 removal capacity is the greatest. This confirms that inducing stable loading of CeNPs through carboxyl modification greatly improves the removal efficiency of reactive oxygen species.

[0183] Figure 33 The blood perfusion system of this invention, as demonstrated in an animal model of severe sepsis, significantly improved survival rates.

[0184] In this experiment, cerium dioxide nanoparticles (CeNPs) were loaded onto sulfonated PS-DVB, and surface-coated microbeads (PS-PS-DVB@Ce) were used for hemoperfusion. The group treated with this system showed a 100% survival rate, a significant difference compared to the 0% survival rate of the control group receiving only standard treatment. These results clearly demonstrate that the S-PS-DVB@Ce-based hemoperfusion system of this invention can effectively reduce mortality from sepsis.

[0185] Figure 34 The hemoperfusion system of this invention, as illustrated in an animal model of severe sepsis, significantly improved hypotension. The X-axis represents the time elapsed since LPS administration, and the Y-axis represents mean arterial pressure (MAP, mmHg). The control group (receiving only standard treatment, dashed line) showed a sustained decrease in mean arterial pressure, a typical phenomenon in refractory septic shock, while the group treated with the hemoperfusion system of this invention (such as perfusion using PVP-coated S-PS / DVB@Ce microbeads, solid line) tended to maintain or increase MAP levels, confirming the effective improvement of hypotension.

[0186] Industrial applicability This invention relates to an extracorporeal blood perfusion system that can effectively remove reactive oxygen species (ROS) that cause inflammation from the blood outside the body, which can help treat sepsis or various inflammatory diseases.

Claims

1. A structure for in vitro blood perfusion, comprising cerium dioxide nanoparticles and a carrier loading the cerium dioxide nanoparticles.

2. The structure for extracorporeal blood perfusion according to claim 1, wherein, The carrier is a polymer-based carrier in which the polymer accounts for more than 50 wt% of the total carrier weight.

3. The structure for extracorporeal blood perfusion according to claim 1, wherein, The carrier comprises one or more polymers selected from the group consisting of chitosan, chitin, polyethylene, polyacrylonitrile, polyvinylidene fluoride, polysulfone, polyethersulfone, polystyrene, polyvinyl alcohol, polymethyl methacrylate, cellulose, or cellulose acetate.

4. The structure for extracorporeal blood perfusion according to claim 1, wherein, The carrier comprises one or more polymers selected from the group consisting of polystyrene (PS), polyethersulfone (PES), and polyacrylonitrile (PAN).

5. The structure for extracorporeal blood perfusion according to claim 1, wherein, The carrier is porous microspheres.

6. The structure for extracorporeal blood perfusion according to claim 1, wherein, The carrier is a porous fiber.

7. The structure for extracorporeal blood perfusion according to claim 1, wherein, The carrier is a membrane carrier.

8. The structure for extracorporeal blood perfusion according to claim 1, wherein, The structure used for in vitro blood perfusion removes reactive oxygen species through the catalytic action of cerium dioxide nanoparticles.

9. The structure for extracorporeal blood perfusion according to claim 5, wherein, The size (diameter) of the porous microspheres is 1μm to 1500μm.

10. The structure for extracorporeal blood perfusion according to claim 5, wherein, The porous microspheres have a pore volume of 0.1 cm³. 3 / g to 13.6cm 3 / g.

11. The structure for extracorporeal blood perfusion according to claim 5, wherein, The surface area of ​​the porous microspheres is 100m². 2 / g to 1000m 2 / g.

12. The structure for extracorporeal blood perfusion according to claim 6, wherein, The diameter of the porous fiber is 10μm~50μm.

13. The structure for extracorporeal blood perfusion according to claim 6, wherein, The porous fiber has a porosity of less than 10%.

14. The structure for extracorporeal blood perfusion according to claim 7, wherein, The membrane carrier has an outer diameter of 200μm~350μm, an inner diameter of 150μm~250μm, a wall thickness of 30μm~100μm, a pore size of 5nm~50nm, and a porosity of 30%~80%.

15. The structure for extracorporeal blood perfusion according to claim 7, wherein, The membrane carrier has a molecular weight cutoff (MWCO) of 10,000 Da to 100,000 Da and an ultrafiltration coefficient (UF coefficient) of 5 mL / h / mmHg / m 2 ~80mL / h / mmHg / m 2 The surface area is 0.2m². 2 ~2.5m 2 .

16. The structure for extracorporeal blood perfusion according to claim 1, wherein, The structure for in vitro blood perfusion contains cerium dioxide nanoparticles at a concentration of 0.5 mg / g or higher.

17. The structure for extracorporeal blood perfusion according to claim 1, wherein, The structure for in vitro blood perfusion is coated with one or more materials selected from the group consisting of PEG (polyethylene glycol) and PVP (polyvinylpyrrolidone).

18. An extracorporeal blood perfusion tube comprising a structure for extracorporeal blood perfusion according to any one of claims 1 to 17.

19. The extracorporeal blood perfusion tube according to claim 18, wherein, The structure used for extracorporeal blood perfusion has a volume fraction of 50% to 90% relative to the entire extracorporeal blood perfusion tube.

20. An extracorporeal blood purification device comprising the extracorporeal blood perfusion tube according to claim 18.

21. An extracorporeal blood purification method, comprising the following steps: (a) Contacting the blood separated from the subject with the structure for extracorporeal blood perfusion according to any one of claims 1 to 17; (b) Using the structure for in vitro blood perfusion to remove reactive oxygen species from the blood; as well as (c) Recover the blood that has been cleared of the reactive oxygen species.

22. A method for preparing a structure for in vitro blood perfusion according to any one of claims 1 to 17, comprising the following steps: (a) Preparing a structure comprising one or more polymers for in vitro blood perfusion; (b) Incorporating cerium dioxide nanoparticles into the structure for in vitro blood perfusion.

23. The method according to claim 22, wherein, Step (a) involves modifying the surface of a structure for in vitro blood perfusion with an agent comprising one or more functional groups selected from the group consisting of amine, sulfonic acid, thiol, carboxylic, hydroxyl, or epoxy groups.

24. The method according to claim 22, wherein, The method further includes coating the surface of the structure used for in vitro blood perfusion with one or more polymers selected from the group consisting of PEG (polyethylene glycol) and PVP (polyvinylpyrrolidone).

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