Therapeutic magnetic biomolecule-metal ion self-assembled complexes

Abstract

To provide a magnetic biomolecule-metal ion self-assembly complex for treatment effective for cancer treatment, osteoarthritis treatment and bone defect treatment.SOLUTION: A magnetic biomolecule-metal ion self-assembly complex for treatment includes an iron ion and at least one ligand, where the ligand and the iron ion are reversibly self-assembled with each other or self-disassembled from each other, where the ligand and the iron ion are self-assembled with each other via a first bond to form a self-assembly, where the self-assembling is performed by at least one of the metal ion and the ligand. There are multiple such self-assemblies such that the self-assemblies adjacent to each other self-bind to each other via a second bond.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a therapeutic magnetic biomolecule-metal ion self-assembly complex, and more particularly to a therapeutic magnetic biomolecule-metal ion self-assembly complex having improved efficacy in the treatment of cancer, osteoarthritis and bone defects. [Background technology]

[0002] Recently, anti-cancer treatments have relied on chemically synthesized drugs. Chemotherapeutic drugs, including doxorubicin, typically used in cancer treatment, treat cancer by inducing apoptosis in cancer cells. Meanwhile, doxorubicin, cisplatin, and methotrexate, three of the most common anti-cancer drugs, are known to cause side effects on the heart, kidneys, and nerves, respectively. These side effects make it difficult to prescribe the appropriate drug depending on the patient's condition. In particular, doxorubicin, which is widely used, has a very narrow therapeutic index (TI), which is the range in which the drug can be used. Using concentrations higher than this TI is known to cause fatal side effects.

[0003] In addition, these chemotherapy drugs can cause serious side effects such as hair loss, bone marrow suppression, vomiting, rash, stomatitis, hypersensitivity, allergy, cardiac damage, injection site injury, radiation recall, and treatment-related leukemia. Furthermore, as cancer progresses, resistance to cell death increases, which can weaken the therapeutic efficacy of chemotherapy drugs.

[0004] Therefore, conventional anticancer drugs have evolved to target cancer cells and induce cell death, and many technologies have been developed. However, new methods are being researched to eliminate cancer cells that are resistant to cell death by conventional drugs while maintaining their effectiveness in eliminating cancer cells and causing fewer side effects.

[0005] Arthritis is one of the most common chronic diseases in the world, but the pathophysiology of osteoarthritis has not been thoroughly studied, and there is currently no complete cure. Osteoarthritis damages articular cartilage, causing severe pain and disability in patients, and physiological regeneration of articular cartilage occurs only to a limited extent. Therefore, various researchers have used animal models to study the pathophysiology of osteoarthritis and search for treatments. Recently, medications have been used to treat osteoarthritis. Medications used to treat arthritis include hyaluronic acid, nonsteroidal anti-inflammatory drugs (NSAIDs), and steroid triamcinolone (TAA), which merely inhibit inflammation, but there are almost no drugs that can regenerate cartilage.

[0006] In addition, various types of cells exist in bones and joints, including mesenchymal stem cells (MSCs), chondrocytes, macrophages, and fibroblasts. When the cytokine balance in the synovial fluid of the joint cavity is disrupted, pro-inflammatory cytokines are increased more than anti-inflammatory cytokines, worsening osteoarthritis.

[0007] Therefore, research is needed into drugs and treatment methods that are effective in treating osteoarthritis and can regulate the cytokine balance within the osteoarthritis to a normal osteoarthritis level.

[0008] Extensive bone defects in long bone fractures, including those of the femur, are clinically challenging to treat. Recently, the induced membrane technique has been used to treat such extensive bone defects. In this technique, after removing all dead and infected bone in a primary surgery, the defect is filled with bone cement such as calcium phosphate. After waiting approximately 4–6 weeks for the formation of an induced membrane, a secondary surgery is performed to remove the bone cement and graft a large amount of autologous bone into the bone defect. BMP, which is used in this induced membrane technique, is prohibitively expensive for practical use in treatment and presents clinical challenges that must be overcome. Furthermore, autologous bone grafts containing BMP are often used in limited cases, resulting in insufficient harvested autologous bone or insufficient bone volume even when all available autologous bone, including the pelvis and tibia, is harvested due to the large size of the bone defect.

[0009] Autologous bone grafts can cause many complications, such as delayed rehabilitation due to pain at the graft site, bleeding, infection, and iatrogenic fractures, and there is a need to research graft materials that can replace autologous bone using an induction membrane method. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] KR10-0901127 B1 Summary of the Invention [Problem to be solved by the invention]

[0011] The object of the present invention is to provide a therapeutic magnetic biomolecule-metal ion self-assembly complex that can self-assemble into various forms based on substances in the body and does not cause side reactions in the body.

[0012] Another object of the present invention is to provide a therapeutic magnetic biomolecule-metal ion self-assembly complex that is non-toxic to living organisms, self-assembles into various forms depending on the type of ligand, and can be easily controlled in position outside the living organism to effectively treat a target location. [Means for solving the problem]

[0013] In accordance with one aspect of the present invention, embodiments of the present invention include therapeutic magnetic biomolecule-metal ion self-assembly complexes.

[0014] In one embodiment, the therapeutic magnetic biomolecule-metal ion self-assembly complex comprises an iron ion; and one or more ligands; wherein the ligand and the iron ion can reversibly self-assemble or self-disassemble, and the ligand and the iron ion can self-assemble through a first bond to form a self-assembly.

[0015] In one embodiment, the self-assembly is formed by one or more of the metal ions and the ligands, and the self-assemblies may be provided in a plurality of pieces, with adjacent self-assemblies being self-bonded by a second bond.

[0016] In one embodiment, the first bond may include a coordinate bond, and the second bond may include one or more of a hydrogen bond and a π-π interaction.

[0017] In one embodiment, the self-assembly or adjacent self-assemblies are self-assembled for a first time or self-disassembled for a second time under physiologically relevant conditions, and the first time may be 1 minute to 24 hours, and the second time may be 1 day to 90 days.

[0018] In one embodiment, the ligand may include at least one of a phosphate and a phosphonate.

[0019] In one embodiment, the ligand may be at least one of AMP, ADP, ATP, TMP, TDP, TTP, CMP, CDP, CTP, GMP, GDP, GTP, UMP, UDP, UTP, DNA, RNA, 2-aminoethylphosphonic acid (AEP), three-nucleotide nucleic acid (TNA), glycol nucleic acid (GNA), 1,5-anhydrohexitol nucleic acid (HNA), 1,5-anhydroatritol nucleic acid (ANA), 2'-deoxy-2'-fluoroarabino nucleic acid (FANA), and cyclohexenyl nucleic acid (CeNA).

[0020] In one embodiment, the ligand may include one or more of AMP (adenosine monophosphate) and ATP (adenosine triphosphate).

[0021] In one embodiment, when the ligand is ATP (adenosine triphosphate), the therapeutic magnetic biomolecule-metal ion self-assembly complex may be provided in the form of individual spheres, and when the ligand is AMP (adenosine monophosphate), the therapeutic magnetic biomolecule-metal ion self-assembly complex may be provided in the form of a three-dimensional aggregate having micropores, formed by aggregating multiple self-assemblies.

[0022] In one embodiment, the self-assembly has paramagnetic properties, and the movement of the self-assembly may be controlled by applying an external magnetic field.

[0023] In one embodiment, the self-assembly may be accelerated to self-decompose under at least one of a condition containing a chelating agent, a strong acid condition, and a strong base condition.

[0024] In the condition containing the chelating agent, the chelating agent may be one or more of EDTA (ethylenediaminetetraacetic acid), bipyridyl, and ferrozine, and the pH of the strong acid condition may be 2 to 5, and the pH of the strong base condition may be 9 to 12.

[0025] In one embodiment, the self-assembly generates excessive reactive oxygen species (ROS) under conditions containing hydrogen peroxide, and the ROS oxidize cellular phospholipids and inhibit GPX4 (glutathione peroxidase 4) or System xc-cystine / glutamate antiporter (Xc), thereby inducing cancer cell death through ferroptosis.

[0026] In one embodiment, the self-assembly is paramagnetic and its movement is controlled by application of an external magnetic field, and application of the external magnetic field may cause the self-assembly to move to target cancer cells and induce the death of the cancer cells through ferroptosis.

[0027] In one embodiment, the self-assembly may not exhibit toxicity to normal cells while inducing cancer cell death through ferroptosis.

[0028] In one embodiment, the self-assembly is used for cancer treatment, and the cancer may be any one or more selected from the group consisting of breast cancer, colorectal cancer, rectal cancer, lung cancer, colon cancer, thyroid cancer, oral cancer, pharyngeal cancer, laryngeal cancer, cervical cancer, brain cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, pancreatic and intestinal cancer, prostate cancer, skin cancer, tongue cancer, uterine cancer, stomach cancer, bone cancer, and blood cancer.

[0029] In one embodiment, the ligand comprises ATP (adenosine triphosphate), and the self-assembly may release the ATP during self-decomposition to promote M2 polarization of macrophages via the P2Y1 receptor.

[0030] In one embodiment, the self-assembly promotes M2 polarization of macrophages distributed in the synovial membrane of bone joints, and the M2 polarization of the macrophages may have anti-inflammatory activity in the synovial fluid of bone joints.

[0031] In one embodiment, the self-assembly can maintain an anti-inflammatory environment in the synovial fluid of bone joints, protect bone and cartilage, and prevent, ameliorate, or treat osteoarthritis.

[0032] In one embodiment, the self-assembly has paramagnetic properties and its movement is controlled by application of an external magnetic field, and application of the external magnetic field causes the self-assembly to move to a target bone joint site, protecting bone and cartilage and preventing, improving or treating osteoarthritis.

[0033] In one embodiment, the self-assembly promotes M2 polarization of macrophages distributed in the synovial membrane of bone joints, and the M2 polarization of the macrophages may have anti-inflammatory activity in the synovial fluid of bone joints without exhibiting toxicity to normal cells.

[0034] In one embodiment, the ligand comprises AMP (adenosine monophosphate), and the therapeutic magnetic biomolecule-metal ion self-assembly complex is provided as a three-dimensional aggregate having micropores by aggregation of multiple self-assemblies, and the three-dimensional aggregate may have an average diameter of 500 μm to 10 cm.

[0035] In one embodiment, the self-assembly may be used as a bone graft material that is transplanted in the form of the three-dimensional aggregate into a bone defect site to repair the missing bone tissue.

[0036] In one embodiment, the self-assembly is paramagnetic and its movement is controlled by the application of an external magnetic field, the self-assembly is transplanted into the bone defect site in the form of the three-dimensional aggregate, and the application of the external magnetic field may cause the self-assembly to move in one or more directions.

[0037] In one embodiment, the self-assembly may be implanted into a bone defect site, and host cells may be attached to the surface of the self-assembly to form a scaffold.

[0038] In one embodiment, the self-assembly has paramagnetic properties, and its movement is controlled by application of an external magnetic field, and host cells attached to the surface of the self-assembly may also move together with the self-assembly by application of the external magnetic field.

[0039] In one embodiment, the self-assembly may be implanted into a bone defect site, and the self-assembly may self-degrade to release the AMP for 1 to 70 days.

[0040] In one embodiment, the AMP released by the self-assembly is degraded to adenosine on the surface of the host cell to promote signaling of adenosine receptors on the surface of the host cell, and the adenosine receptors may include adenosine A2B receptors.

[0041] In one embodiment, the AMP released by the self-assembly may promote osteogenic differentiation of mesenchymal stem cells (MSCs) by signaling adenosine receptors in the MSCs, and the osteogenic differentiation of the MSCs may promote bone formation.

[0042] In one embodiment, the self-assembly may not exhibit toxicity to normal cells while being transplanted into a bone defect site to regenerate the missing bone tissue.

[0043] In one embodiment, the ligand comprises one or more of AMP (adenosine monophosphate) and ATP (adenosine triphosphate), the self-assemblies are paramagnetic, and their movement is controlled by an externally applied magnetic field. When the ligand is one or more of AMP and ATP, the self-assemblies induce the death of cancer cells through ferroptosis. When the ligand is ATP, the self-assemblies prevent, ameliorate, or treat osteoarthritis. When the ligand is AMP, the self-assemblies can be transplanted into a bone defect site to regenerate the missing bone tissue. [Effects of the Invention]

[0044] According to the present invention described above, it is possible to provide a therapeutic magnetic biomolecule-metal ion self-assembly complex that is effective in cancer treatment by enabling targeted treatment at a local site, having no side effects, and easily inducing cell death.

[0045] Furthermore, the present invention provides a therapeutic magnetic biomolecule-metal ion self-assembly complex that regulates the cytokine balance in osteoarthritis-affected bone joints to a normal bone joint level in order to prevent the progression of osteoarthritis.

[0046] Furthermore, the present invention can provide a therapeutic magnetic biomolecule-metal ion self-assembly complex that effectively treats bone defects and has no side effects. [Brief explanation of the drawings]

[0047] [Figure 1] 1 is a diagram illustrating a state in which iron ions and ATP form a self-assembly according to an embodiment of the present invention. [Figure 2] 1 is a diagram illustrating a state in which iron ions and AMP form a self-assembly according to an embodiment of the present invention. [Figure 3] This shows an SEM image of Fe-ATP self-assembly and the results of measuring paramagnetism. [Figure 4]1A-1C are SEM images of Fe-ATP self-assemblies with average diameters of 150 nm and 70 nm, respectively, according to one embodiment of the present invention. [Figure 5] TEM image of Fe-AMP self-assembly. [Figure 6] 1 is a diagram illustrating the induction of ferroptosis in cancer cells using Fe-ATP self-assembly. [Figure 7] 1 is a graph showing the amount of iron ion released from Fe-ATP self-assemblies at pH 5.5 or pH 7.4, measured using an iron assay kit. [Figure 8] The intracellular ROS concentration measured by DCFDA staining under each condition is shown. [Figure 9] This is the result of analyzing the IVIS L-012 ROS signal. [Figure 10] The results confirmed ferroptosis of cancer cells due to sustained Fe2+ release from magnetically targeted Fe-ATP self-assemblies. [Figure 11] We have shown that magnetically targeted Fe-ATP self-assembly effectively treats osteosarcoma cancer in a manner similar to doxorubicin. [Figure 12] This is the result of confirming the toxicity of Fe-ATP self-assemblies after transplantation into a living body. [Figure 13] This result confirms the chondroprotective effect of magnetically targeted Fe-ATP self-assemblies. [Figure 14] 1 is a graph showing the cumulative ATP release profile of Fe-ATP self-assemblies measured by HPLC. [Figure 15] This is the result of confirming the polarization of macrophages due to the disassembly of Fe-ATP self-aggregates using immunofluorescence images. [Figure 16] This is the result of flow cytometry confirming the polarization of macrophages due to the disassembly of Fe-ATP self-aggregates. [Figure 17]The disassembly of Fe-ATP self-assembly was confirmed by Western blotting. [Figure 18] C-Arm and 3-D micro-CT images of a knee joint to confirm the therapeutic effect of Fe-ATP self-assembly on osteoarthritis. [Figure 19] 2-D micro-CT images of the knee joint of an osteoarthritis model at 8 weeks, H&E stained images, and safranin-o (SO) stained images. [Figure 20] These are H&E stained, safranin-o (SO) stained, and collagen X stained images of an 8-week osteoarthritis model. [Figure 21] 10 shows images of iNOS and Arg-1 staining in the synovial membrane of the knee joint of an osteoarthritis model at 8 weeks. [Figure 22] 1 shows the toxicity results of Fe-ATP self-assembly confirmed in a rat model of osteoarthritis. [Figure 23] 1 is a diagram showing the bone regeneration effect of 3D Fe-AMP self-assembly. [Figure 24] This is an SEM image showing the micropore structure formed by the aggregation of 3D Fe-AMP self-assemblies. [Figure 25] 1 is a diagram showing the magnetic targeting of 3D Fe-AMP self-assemblies. [Figure 26] 1 is a graph showing AMP released over time from Fe-AMP self-assemblies. [Figure 27] Immunofluorescence imaging confirmed that AMP-degrading adenosine released by 3D Fe-AMP self-assemblies promoted osteogenic differentiation. [Figure 28] Western blotting results confirming osteogenic differentiation by CD73-mediated AMP-cleaving adenosine released by 3D Fe-AMP self-assemblies. [Figure 29] C-Arm images of the femur to confirm fracture at 0, 2, 4, 6 and 8 weeks. [Figure 30]Micro-CT images of femurs at 8 weeks to identify fractures. [Figure 31] H&E, osteocalcin, and TRAP staining images of the fracture. [Figure 32] These results confirm the toxicity of 3D Fe-AMP self-assemblies in vivo. MODE FOR CARRYING OUT THE INVENTION

[0048] Other specific details of the embodiments are included in the detailed description and drawings.

[0049] The advantages and features of the present invention, as well as methods for achieving them, will become clearer with reference to the embodiments described below in detail with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below and may be embodied in a variety of different forms. Unless otherwise specified in the following description, all numbers, values, and / or expressions expressing components, reaction conditions, and component contents in the present invention are approximate values ​​that inherently reflect various uncertainties in measurement that arise in obtaining such values, among others, and should be understood to be modified in all cases by the term "about." Furthermore, when a range of values ​​is disclosed herein, such range is continuous and includes all values ​​from the minimum value to the maximum value, inclusive, unless otherwise specified. Furthermore, when such a range refers to integers, it includes all integers from the minimum value to the maximum value, inclusive, unless otherwise specified.

[0050] Additionally, when a range is described for a variable herein, the variable should be understood to include all values ​​within the described range, including the recited endpoints of the range. For example, the range "5 to 10" should be understood to include not only the values ​​5, 6, 7, 8, 9, and 10, but also any subranges such as 6 to 10, 7 to 10, 6 to 9, and 7 to 9, as well as any value between the integers within the described range, such as 5.5, 6.5, 7.5, 5.5 to 8.5, and 6.5 to 9. For example, the range "10% to 30%" should be understood to include values ​​such as 10%, 11%, 12%, 13%, and all integers up to and including 30%, as well as any subranges such as 10% to 15%, 12% to 18%, and 20% to 30%, as well as any value between the integers within the described range, such as 10.5%, 15.5%, 25.5%, etc.

[0051] Figure 1 is a schematic diagram illustrating the self-assembly of iron ions and ATP according to one embodiment of the present invention, and Figure 2 is a schematic diagram illustrating the self-assembly of iron ions and AMP according to one embodiment of the present invention.

[0052] 1 and 2, a therapeutic magnetic biomolecule-metal ion self-assembly complex according to this embodiment may include a therapeutic magnetic biomolecule-metal ion self-assembly complex that includes iron ions and one or more ligands, where the ligands and iron ions reversibly self-assemble or self-disassemble, and the ligands and iron ions self-assemble through a first bond to form a self-assembly. Furthermore, in the therapeutic magnetic biomolecule-metal ion self-assembly complex, the self-assembly may be formed by self-assembly through one or more of the metal ions and the ligands, and a plurality of the self-assemblies may be provided, with adjacent self-assemblies self-bonded through a second bond.

[0053] In this embodiment, the therapeutic magnetic biomolecule-metal ion self-assembly complex (hereinafter referred to as biomolecule-metal ion self-assembly complex) is effective in treating diseases, has magnetic properties, and includes a self-assembly formed by the self-assembly of biomolecules and metal ions, and the self-assembly can be provided in one or more forms to form a complex.

[0054] The ligand may include at least one of phosphate and phosphonate. Specifically, the ligand may be at least one of AMP, ADP, ATP, TMP, TDP, TTP, CMP, CDP, CTP, GMP, GDP, GTP, UMP, UDP, UTP, DNA, RNA, 2-aminoethylphosphonic acid (AEP), three-nucleotide nucleic acid (TNA), glycol nucleic acid (GNA), 1,5-anhydrohexitol nucleic acid (HNA), 1,5-anhydroatritol nucleic acid (ANA), 2'-deoxy-2'-fluoroarabino nucleic acid (FANA), and cyclohexenyl nucleic acid (CeNA). More specifically, the ligand may include at least one of adenosine monophosphate (AMP) and adenosine triphosphate (ATP).

[0055] The ligand and the iron ion can self-bond to each other through a first bond to form a self-assembly, and adjacent self-assemblies can self-bond to each other through a second bond to form a biomolecule-metal ion self-assembly complex. In the biomolecule-metal ion self-assembly complex, the first bond may include a coordinate bond, and the second bond may include at least one of a hydrogen bond and a π-π interaction.

[0056] In Figure 1, iron ions (Fe 2+) can self-assemble with the ligand ATP through coordination bonds to form Fe-ATP self-assemblies. The Fe-ATP self-assemblies can self-assemble through one or more of hydrogen bonds and π-π interactions between adjacent Fe-ATP self-assemblies to form a therapeutic magnetic biomolecule-metal ion self-assembly complex according to one embodiment of the present invention.

[0057] Also, in Figure 2, iron ions (Fe 2+ ) can self-assemble with the ligand AMP through coordination bonds to form Fe-AMP self-assemblies. The Fe-AMP self-assemblies can self-assemble through one or more of hydrogen bonds and π-π interactions between adjacent Fe-AMP self-assemblies to form a biomolecule-metal ion self-assembly complex according to one embodiment of the present invention.

[0058] The iron ion can form a metal complex by coordinate bonding with the phosphate group of ATP or AMP and water (HO). The aromatic ring of the adenine moiety of ATP or AMP can form a π-π interaction. The nitrogen of the adenine moiety can form a hydrogen bond with a water molecule bound to the iron ion. The biomolecule-metal ion self-assembly complex can self-bond using one or more of a coordinate bond, a π-π interaction, and a hydrogen bond, forming a complex containing one or more self-assemblies.

[0059] The self-assembly or adjacent self-assemblies may be self-assembled for a first time or self-disassembled for a second time under physiologically relevant conditions, where the first time may be 1 minute to 24 hours, and the second time may be 1 day to 90 days.

[0060] The physiological conditions refer to temperature, pH, osmotic pressure, ionic strength, viscosity, and similar biochemical parameters that are compatible with living organisms and / or are typically present in living cultured yeast cells or mammalian cells. For example, in the case of human cells, the physiological conditions include a temperature of about 37°C, e.g., a temperature in the range of 35-39°C, a pressure in the range of 1 kPa-200 kPa, and a pH of about 7. Specifically, the physiological conditions refer to conditions that are not toxic to living organisms or animals and can be maintained by living organisms or animals.

[0061] The self-assembly can be completed in a first time period of 1 minute to 24 hours, but if the time period is less than 1 minute, the self-assembly cannot stably form the first bond or the second bond, which is problematic, and if the time period is 24 hours, the self-assembly is completed sufficiently, so maintaining the time period longer than this period reduces efficiency. Specifically, the first time period may be about 1 minute to 20 hours, about 1 minute to 15 hours, about 1 minute to 10 hours, about 1 minute to 5 hours, about 1 minute to 2 hours, or about 1 minute to 30 minutes.

[0062] The self-assembly can be decomposed for a second time period of 1 day to 90 days, and can exhibit a stable therapeutic effect by releasing iron ions or ligands during the decomposition period. Specifically, the second time period can be about 1 day to 45 days, about 1 day to 30 days, about 1 day to 15 days, or about 1 day to 10 days.

[0063] When the ligand is ATP (adenosine triphosphate), the therapeutic magnetic biomolecule-metal ion self-assembly complex may be provided in the form of individual spheres, or when the ligand is AMP (adenosine monophosphate), the therapeutic magnetic biomolecule-metal ion self-assembly complex may be provided in the form of a three-dimensional aggregate having micropores, formed by aggregating a plurality of self-assemblies.

[0064] Specifically, when the ligand is ATP, the biomolecule-metal ion self-assembly complex may be spherical and have a uniform diameter. The diameter of the biomolecule-metal ion self-assembly complex may vary depending on one or more of the concentration of the ligand and the concentration of iron ions. Specifically, when the concentration of iron ions is high, the diameter of the self-assembly may increase. Furthermore, when the concentration of the ligand is high, the diameter of the self-assembly may increase. When the ligand of the biomolecule-metal ion self-assembly complex according to this embodiment is ATP, the diameter of the biomolecule-metal ion self-assembly complex can be controlled by controlling the concentration of iron ions and / or the concentration of ATP.

[0065] When the ligand is AMP, the biomolecule-metal ion self-assembly complex may be in the form of a three-dimensional aggregate. Specifically, it may be in the form of a volume having a plurality of micropores inside. For example, even when the ligand of the biomolecule-metal ion self-assembly complex is AMP, the size of the three-dimensional aggregate can be controlled by controlling the concentration of the iron ions and / or the concentration of AMP. Specifically, the size of the three-dimensional aggregate can be increased by increasing the concentration of iron ions or the concentration of AMP. The size of the three-dimensional aggregate may be 100 nm to 100 cm. Specifically, the size of the three-dimensional aggregate may be the average diameter in the case of a sphere, the average diameter and height of the cross section in the case of a cylinder, or the average side length and height in the case of a polygon. The size of the three-dimensional aggregate may be about 100 nm to 80 cm, or about 100 nm to 50 cm, or about 100 nm to 30 cm, or about 100 um to 30 cm, or about 200 um to 30 cm, or about 300 um to 30 cm, or about 400 um to 30 cm, or 500 um to 30 cm, or 500 um to 20 cm, or 500 um to 10 cm.

[0066] The self-assembly complexes are paramagnetic, and the movement of the self-assemblies can be controlled by applying an external magnetic field. Specifically, the biomolecule-metal ion self-assembly complexes are provided in vivo or ex vivo, and the movement of the self-assemblies constituting the biomolecule-metal ion self-assembly complexes can be controlled by applying an external magnetic field. Therefore, the biomolecule-metal ion self-assembly complexes according to the present embodiment can be controlled to move to a desired location. Specifically, even after injection into a living body, the biomolecule-metal ion self-assembly complexes can be moved to target cells or tissues, thereby exerting a direct effect.

[0067] The self-assembly may be accelerated to self-decompose under at least one of a condition containing a chelating agent, a strong acid condition, and a strong base condition.

[0068] When the chelating agent is contained, the chelating agent may be one or more of EDTA (ethylenediaminetetraacetic acid), bipyridyl, and ferrozine.

[0069] The chelating agent can form a bond with the iron ions instead of the ligand, thereby promoting the self-disassembly of the self-assembly. The chelating agent may be a substance having a higher affinity for the iron ions than the ligand, such as EDTA.

[0070] The pH of the strong acidic conditions may be 2 to 5, and the pH of the strong basic conditions may be 9 to 12. Specifically, the pH of the strong acidic conditions may be 3 to 4, and the pH of the strong basic conditions may be 10 to 11. For example, the strong acid may be hydrochloric acid (HCl), and the strong base may be NaOH, for example.

[0071] The strong acid or strong base conditions can promote the breakdown of the bond between the iron ion and the ligand, and can also promote the self-disassembly of the self-assembly complex.

[0072] The self-assembly may be paramagnetic. As the volume of the self-assembly or biomolecule-metal ion self-assembly complex increases, the magnetic moment may increase. Therefore, the larger the self-assembly complex, the stronger the attractive (or repulsive) force of the magnetic field. Specifically, when a magnetic field is applied to the biomolecule-metal ion self-assembly complex in vivo and in vitro, the movement of the biomolecule-metal ion self-assembly complex may be controlled by the magnetic field. The larger the biomolecule-metal ion self-assembly complex, the greater the movement speed of the biomolecule-metal ion self-assembly complex. Furthermore, the biomolecule-metal ion self-assembly complex according to this embodiment becomes magnetic only when a magnetic field is applied to the biomolecule-metal ion self-assembly complex, allowing for easy manipulation of the biomolecule-metal ion self-assembly complex in vivo and in vitro.

[0073] The biomolecule-metal ion self-assembly complex according to this embodiment can induce ferroptosis and kill specific cancer cells.

[0074] Anti-cancer treatments typically rely on chemically synthesized drugs, and representative drugs such as doxorubicin, cisplatin, and methotrexate cause side effects on the heart, kidneys, and nerves, which can be problematic. In addition, targeted therapies and immunotherapies that target cancer have been developed and used to reduce damage to normal tissues during cancer treatment. However, in the case of sarcoma, targeted therapies are difficult to use because each patient has different target substances, and immunotherapies are expensive, making them extremely difficult to use in actual treatment.

[0075] Meanwhile, the biomolecule-metal ion self-assembly complex according to this embodiment is non-toxic to living organisms and can target and migrate cancer cells using externally applied magnetism. Furthermore, the biomolecule-metal ion self-assembly complex can effectively kill cancer cells by inducing ferroptosis in the cancer cells, without affecting surrounding normal cells and without toxicity in vivo, making it stable for use. Ferroptosis can induce cell death in a manner different from cell death induced by oxidative damage to cellular phospholipids.

[0076] In the biomolecule-metal ion self-assembly complex according to this embodiment, the self-assemblies generate excessive amounts of reactive oxygen species (ROS) in the presence of hydrogen peroxide at a concentration of 10 μM or more, and the amount of reactive oxygen species generated is proportional to the concentration of the added self-assemblies and the concentration of hydrogen peroxide. The amount of reactive oxygen species generated can be four times or more greater than that of cancer cells before the addition of the self-assemblies and hydrogen peroxide, and the reactive oxygen species can effectively induce cancer cell death through ferroptosis. Specifically, the concentration of hydrogen peroxide can be about 10 μM to 10 M, or 10 μM to 5 M, or 20 μM to 10 M, or 30 μM to 10 M, or 40 μM to 10 M, or 50 μM to 10 M.

[0077] Generally, normal cells have a hydrogen peroxide concentration of about 20 nM or less, while cancer cells have a hydrogen peroxide concentration of about 10 μM or more, for example, 10 μM to 100 μM, which is higher than normal cells. Therefore, the biomolecule-metal ion self-assembly complex according to this embodiment is self-decomposed only in cancer cells that exhibit a high hydrogen peroxide concentration, and produces Fe. 2+ and the released Fe 2+ The Fenton reaction generates excessive amounts of ROS, which can lead to cancer cell death through ferroptosis.

[0078] These reactive oxygen species oxidize cellular phospholipids and inhibit glutathione peroxidase 4 (GPX4) or System xc-cystine / glutamate antiporter (Xc), which can induce cancer cell death through ferroptosis. GPX4 is an enzyme present in cells that restores oxidized phospholipids and is regulated by System xc-cystine / glutamate antiporter (Xc).

[0079] The biomolecule-metal ion self-assembly complex according to the present embodiment includes iron ions, which can be released by autolysis of the self-assemblies that constitute the biomolecule-metal ion self-assembly complex. The iron ions are biological substances that react with hydrogen peroxide, which is present at high concentrations in cancer cells, to generate ROS through a Fenton reaction. The ROS can damage cellular phospholipids through an oxidation reaction, thereby inducing ferroptosis in the cancer cells. Specifically, the ROS oxidize cellular phospholipids, resulting in the excessive generation of oxidized phospholipids, which reduces GPX4 and Xc. When the excessive generation of oxidized phospholipids makes it difficult for GPX4 to restore them, ferroptosis is induced in the cells.

[0080] If the ferroptosis of these cells affects organs other than cancer cells, cardiac side effects (such as cardiomyopathy) may occur. Therefore, in the process of treating cancer by inducing ferroptosis using artificially synthesized drugs such as doxorubicin, side effects may also occur in normal cells surrounding the cancer cells, which is problematic.

[0081] Meanwhile, the biomolecule-metal ion self-assembly complex according to this embodiment can target a localized area using an externally applied magnetic field, thereby selectively eliminating only cancer cells. The biomolecule-metal ion self-assembly complex is composed of a biomolecule and an iron ion, which is non-toxic to the body, and is therefore non-toxic, has no side effects, and can be used continuously for a long period of time. Furthermore, the biomolecule-metal ion self-assembly complex specifically induces ferroptosis in cancer cells, killing them while leaving normal cells unaffected, making it effective for cancer treatment.

[0082] In the biomolecule-metal ion self-assembly complex, the self-assembly can increase its rate of self-decomposition under acidic conditions. Therefore, while the decomposition rate is slow in normal cells under neutral conditions, the decomposition rate can be accelerated in cancer cells under acidic conditions. Furthermore, cancer cells have relatively higher levels of hydrogen peroxide than normal cells, and the biomolecule-metal ion self-assembly complex can specifically generate ROS under acidic conditions. In other words, in the biomolecule-metal ion self-assembly complex according to this embodiment, the decomposition rate of the self-assembly occurs much faster in cancer cells than in normal cells. Therefore, the decomposition of the iron ions and the ROS-generating Fenton reaction generated by the self-decomposition of the self-assembly act mostly on the cancer cells, with little effect on the normal cells.

[0083] Furthermore, compared to doxorubicin, a typical drug used in conventional treatments, the biomolecule-metal ion self-assembly complex according to this embodiment has the advantages of being able to target localized sites, having fewer side effects, having similar efficacy in killing cancer cells, and not causing cardiac side effects.

[0084] In the biomolecule-metal ion self-assembly complex according to this embodiment, the self-assembly is paramagnetic and its movement is controlled by application of an external magnetic field. When the external magnetic field is applied, the self-assembly moves to target cancer cells and can induce the death of the cancer cells through ferroptosis.

[0085] When the self-assembly induces the death of cancer cells through ferroptosis, it can kill the cancer cells at a level similar to that of doxorubicin. Furthermore, the self-assembly may not exhibit toxicity to normal cells while inducing the death of cancer cells through ferroptosis.

[0086] In the biomolecule-metal ion self-assembly complex according to this embodiment, the self-assembly may be used for cancer treatment, and the cancer may be any one or more selected from the group consisting of breast cancer, colon cancer, rectal cancer, lung cancer, colon cancer, thyroid cancer, oral cancer, pharyngeal cancer, laryngeal cancer, cervical cancer, brain cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, pancreatic and intestinal cancer, prostate cancer, skin cancer, tongue cancer, uterine cancer, stomach cancer, bone cancer, and blood cancer.

[0087] In another embodiment of the biomolecule-metal ion self-assembly complex according to the present invention, the self-assemblies may be formed by self-assembly of iron ions and a ligand via a first bond, and adjacent self-assemblies may self-assemble via a second bond. The ligand may contain adenosine triphosphate (ATP), and the self-assemblies may release the ATP during autolysis to promote M2 polarization of macrophages via the P2Y1 receptor. Furthermore, the self-assemblies may promote M2 polarization of macrophages distributed in the synovial membrane of bone joints, and the M2 polarization of macrophages may have anti-inflammatory activity in synovial fluid of bone joints.

[0088] Arthritis treatment is usually focused on preventing inflammation, and there are almost no drugs that can regenerate cartilage. Various types of cells exist in bones and joints, including MSCs, chondrocytes, macrophages, and fibroblasts. If the cytokine balance in the synovial fluid of the joint cavity is disrupted, pro-inflammatory cytokines that promote inflammation increase more than anti-inflammatory cytokines, worsening osteoarthritis.

[0089] The self-assembly contains the biological substance ATP as a ligand, and the ATP can bind to various cell membrane receptors to regulate cell function. The type of cell membrane receptor that binds to ATP can be controlled by the ATP concentration. The macrophages may have a significant impact on the cytokine balance in intra-articular synovial fluid. Osteoarthritis-related synovial fluid contains approximately 0.5 nM ATP. When the ATP concentration is slightly increased (e.g., several μM) through the Fe-ATP self-assembly, it promotes M2 polarization through the P2Y receptor series and promotes the secretion of anti-inflammatory cytokines. However, when the ATP concentration is very high (e.g., several mM), it promotes M1 polarization through the P2X receptor series and promotes the secretion of pro-inflammatory cytokines.

[0090] Specifically, when the biomolecule-metal ion self-assembly complex according to this embodiment is injected into a site where osteoarthritis has occurred, the self-assemblies decompose to release ATP. The ATP, controlled at a concentration of 5-10 μM, promotes M2 polarization of macrophages distributed in the synovial membrane of bone joints. The M2 polarization of macrophages can have anti-inflammatory activity in the synovial fluid of bone joints. Furthermore, when the ATP generated by the autolysis of the self-assemblies controls the M2 polarization of macrophages, it may have anti-inflammatory activity in the synovial fluid of bone joints without exhibiting toxicity to normal cells. Furthermore, the self-assemblies maintain an anti-inflammatory environment in the synovial fluid of bone joints, protecting bone and cartilage, thereby preventing, ameliorating, or treating osteoarthritis.

[0091] The self-assembly has paramagnetic properties, and its movement can be controlled by applying an external magnetic field. Therefore, after the biomolecule-metal ion self-assembly complex is injected into a living body, the self-assembly moves to the target bone joint site by applying an external magnetic field, thereby protecting the bone and cartilage, and preventing, improving, or treating osteoarthritis.

[0092] According to another embodiment of the present invention, a biomolecule-metal ion self-assembly complex includes one or more self-assemblies, and the self-assemblies may be self-assembled by a first bond between iron ions and a ligand. The ligand may include AMP (adenosine monophosphate), and the biomolecule-metal ion self-assembly complex may be formed by aggregation of a plurality of self-assemblies into a three-dimensional aggregate having micropores. The three-dimensional aggregate may have an average diameter of 500 μm to 10 cm. The self-assembly may be used as a bone graft material by being implanted in the form of a three-dimensional aggregate to repair missing bone tissue. For example, if the average diameter of the biomolecule-metal ion self-assembly complex formed into the three-dimensional aggregate is less than 500 μm, it is too small for use as a bone graft material, which is problematic. If the average diameter exceeds 10 cm, the strength of the three-dimensional aggregate may be reduced, which is problematic.

[0093] The three-dimensional aggregate may have various three-dimensional structures depending on the mold used, and may contain a plurality of micropores therein. For example, the three-dimensional aggregate may have a network structure in which the micropores are three-dimensionally interconnected.

[0094] Long bone fractures, including those of the femur, that result in extensive bone loss are clinically very difficult to treat. Currently, autologous bone grafts and various bone substitutes, such as allogeneic bone, xenogeneic bone, and synthetic bone, are used. However, the higher the proportion of bone substitute, the higher the bone resorption rate, resulting in a lower therapeutic effect. Furthermore, autologous bone grafts can cause numerous complications, such as delayed rehabilitation due to pain at the donor site, bleeding, infection, and iatrogenic fractures.

[0095] In the biomolecule-metal ion self-assembly complex according to an embodiment of the present invention, the self-assembly can self-decompose to release AMP. The released AMP promotes osteogenic differentiation of mesenchymal stem cells (MSCs) by signaling adenosine receptors of MSCs, and the osteogenic differentiation of MSCs can promote bone formation.

[0096] When the biomolecule-metal ion self-assembly complex is implanted into a bone defect site, it acts as a graft material for the bone defect site while simultaneously releasing AMP through the self-decomposition of the self-assembly. The AMP released by the self-assembly can fill the bone defect site and promote MSC attachment and osteogenic differentiation of MSCs.

[0097] Furthermore, the self-assemblies according to this embodiment are paramagnetic, and their movement can be controlled by the application of an external magnetic field. When the self-assemblies are implanted into a bone defect site in the form of three-dimensional aggregates, the self-assemblies can move in one or more directions by the application of the external magnetic field.

[0098] The self-assemblies can be implanted into a bone defect site, and host cells can adhere to the surface of the self-assemblies to form a skeleton. Furthermore, the self-assemblies are paramagnetic, and their migration can be controlled by applying an external magnetic field. For example, when the self-assemblies are implanted into a bone fusion site, they can migrate by applying the external magnetic field. At this time, the host cells attached to the surface of the self-assemblies can also migrate.

[0099] The self-assemblies are implanted into a bone defect site, and the self-assemblies can self-degrade to release the AMP over a period of 1 to 70 days. The AMP released by the self-assemblies can be degraded into adenosine on the surface of host cells to promote signaling from adenosine receptors on the surface of the host cells. For example, the adenosine receptor can include an adenosine A2B receptor.

[0100] When the self-assemblies are implanted into a bone defect site to regenerate the missing bone tissue, they can promote bone regeneration at a level similar to that of calcium phosphate cement, a bone defect implant material. Furthermore, the self-assemblies may not exhibit toxicity to normal cells while implanted into a bone defect site to regenerate the missing bone tissue.

[0101] The therapeutic magnetic biomolecule-metal ion self-assembly complex according to this embodiment may be a self-assembly complex formed by the binding of monomeric self-assemblies, and the self-assemblies may include iron ions and ligands that self-assemble with the iron ions through first bonds. Furthermore, the self-assemblies may be formed by the self-assembly of adjacent self-assemblies through second bonds to form the therapeutic magnetic biomolecule-metal ion self-assembly complex.

[0102] The self-assembly can be decomposed by the surrounding environment of the self-assembly, and the rate of decomposition can be controlled.

[0103] The ligand may include at least one of AMP (adenosine monophosphate) and ATP (adenosine triphosphate). For example, when the self-assembly is self-decomposed, the iron ion or the ligand may be released. Furthermore, the rate at which the iron ion or the ligand is released can be controlled by controlling the rate at which the self-assembly is self-decomposed.

[0104] The size of the therapeutic magnetic biomolecule-metal ion self-assembly complex can be controlled by controlling the manufacturing method, for example, the concentration of the iron ion-containing material and the concentration of the ligand-containing material.

[0105] The self-assembly has paramagnetic properties and its movement can be controlled by an externally applied magnetic field. The movement of the biomolecule-metal ion self-assembly complex can be controlled outside the living body, and even when the biomolecule-metal ion self-assembly complex is transplanted into the living body, the position and movement speed of the biomolecule-metal ion self-assembly complex can be controlled by an externally applied magnetic field.

[0106] When the ligand is one or more of AMP and ATP, the self-assemblies induce cancer cell death through ferroptosis, and when the ligand is ATP, the self-assemblies can prevent, ameliorate, or treat osteoarthritis.Furthermore, when the ligand is AMP, the self-assemblies can be transplanted into a bone defect site to regenerate the missing bone tissue.

[0107] Examples and comparative examples of the present invention are described below. However, the following examples are merely preferred examples of the present invention, and the scope of the present invention is not limited to the following examples.

[0108] Fabrication of self-assemblies 1.Material Adenosine-5'-monophosphate disodium salt (CAS: 4578-31-8, Alfa Aesar) Adenosine 5'-triphosphate disodium salt (CAS: 51963-61-2, Daejung Reagent) Iron(II) chloride (97%, Sigma-Aldrich, 25 g, cat. No. 372870) PMMA (poly(methyl methacrylate), diameter 125~150μm, Bangs Laboratories, BB05N) 2. Preparation of Fe-ATP Self-assemblies Manufacturing Example 1 To synthesize Fe-ATP self-assemblies, FeCl2 solution and ATP (adenosine triphosphate) solution were prepared using deionized water (DI water). FeCl2 solution was prepared by adding 25.35 mg of iron(II) chloride (97%, Sigma-Aldrich, 25 g, cat. No. 372870) to 10 mL of DI water. ATP solution was prepared by adding 110.23 mg of adenosine 5'-triphosphate disodium salt (Adenosine 5'-triphosphate disodium salt, CAS: 51963-61-2, Daejung Reagent) to 10 mL of DI water.

[0109] 1 mL of 20 mM FeCl2 solution was added to a reactor containing 1 mL of 20 mM ATP solution and mixed using a vortex mixer (Vortex Genie 2, Scientific Industries) to prepare a mixed solution. The prepared mixed solution was kept at room temperature for 12 hours to obtain the FeCl2 solution. 2+ The Fe-ATP self-assembly was produced by inducing spontaneous assembly between Fe-ATP and ATP. The solution containing the Fe-ATP self-assembly was centrifuged at 10,000 rpm for 5 minutes. To remove unreacted reagents, the supernatant was discarded, deionized water was added, and the solution was centrifuged twice. After washing, 112.6 mg of particulate Fe-ATP self-assembly was dispersed in 2 mL of deionized water. The produced Fe-ATP self-assembly is shown in Table 1.

[0110] Manufacturing Example 2 The Fe-ATP self-assemblies were prepared in the same manner as in Preparation Example 1, except that 1 mL of 1 mM FeCl2 solution was added to a reactor containing 1 mL of 1 mM ATP solution, and the prepared mixture was reacted at room temperature for 30 minutes. The results are shown in Table 1.

[0111] Manufacturing Example 3 The Fe-ATP self-assemblies were prepared in the same manner as in Preparation Example 1, except that 1 mL of 0.1 mM FeCl solution was added to a reactor containing 1 mL of 0.1 mM ATP solution, and the prepared mixture was reacted at room temperature for 30 minutes. The results are shown in Table 1.

[0112] [Table 1] 3. Preparation of Fe-AMP Self-assemblies Production Example 4 To synthesize Fe-AMP self-assemblies, FeCl2 solution and AMP (adenosine monophosphate) solution were prepared in deionized water (DI water). The FeCl2 solution was prepared by adding 25.35 mg of iron(II) chloride (97%, Sigma-Aldrich, 25 g, cat. No. 372870) to 10 mL of DI water, and the AMP solution was prepared by adding 78.24 mg of AMP (adenosine monophosphate) to 10 mL of DI water.

[0113] 1 mL of 20 mM FeCl2 solution was added to a reactor containing 1 mL of 20 mM AMP solution and mixed using a vortex mixer (Vortex Genie 2, Scientific Industries) to prepare a mixed solution. The prepared mixed solution was kept at room temperature for 12 hours, and the Fe 2+The Fe-AMP self-assembly was produced by inducing spontaneous assembly between Fe-AMP and AMP. The solution containing the Fe-AMP self-assembly was centrifuged at 10,000 rpm for 5 minutes. To remove unreacted reagents, the supernatant was discarded, deionized water was added, and the solution was centrifuged twice. After washing, 89.24 g of particulate Fe-AMP self-assembly was dispersed in 2 mL of deionized water.

[0114] 4.Fe3O4 synthesis Manufacturing Example 5 A mixture was prepared by adding 5.4 g of iron(III) chloride hexahydrate (FeCl3·6H2O) and 18.3 g of sodium oleate to a solvent mixture consisting of 30 mL of deionized water (DI), 40 mL of ethanol (EtOH), and 70 mL of hexane. This mixture was heated at 60°C for 8 hours, and the oleic acid-iron complex contained in the upper hexane layer was washed with DI water. The hexane was then evaporated to obtain the dried oleic acid-iron complex. The oleic acid-iron complex was then mixed with 0.14 g of oleic acid and 5 g of 1-octadecene. This mixture was vigorously stirred at 320°C for 30 minutes and then cooled to 25°C. The Fe3O4 nanoparticles synthesized in the cooled mixture were repeatedly washed with ethanol and centrifuged to produce Fe3O4 nanoparticles with an average diameter of 10 nm.

[0115] Basic performance evaluation method for self-assembly 1. Confirmation of Fe or ATP release characteristics in Fe-ATP self-assembly Using the Fe-ATP self-assembly, the Fe and ATP release properties were confirmed.

[0116] FeCl2 solution was prepared by adding 25.35 mg of iron(II) chloride (Iron(II) chloride, 97%, Sigma-Aldrich, 25 g, cat. No. 372870) to 10 mL of deionized water, and ATP solution was prepared by adding 110.23 mg of adenosine 5'-triphosphate disodium salt (Adenosine 5'-triphosphate disodium salt, CAS: 51963-61-2, Daejung's Reagent) to 10 mL of deionized water.

[0117] 5 mL of 20 mM ATP solution and 5 mL of 20 mM FeCl2 solution were added to a Falcon tube and mixed for 1 minute using a vortex mixer (Vortex Genie2, Scientific Industries) to prepare a mixed solution. The resulting mixed solution was kept at room temperature for 12 hours and then centrifuged at 10,000 rpm for 5 minutes. The supernatant containing unreacted reagents was removed, and 10 mL of deionized water was added. After adding deionized water, the mixture was centrifuged again at 10,000 rpm for 5 minutes and the supernatant was removed to recover the solid Fe-ATP self-assemblies. The removed supernatant was used to measure the ATP loading efficiency by subtracting the amount of ATP unloaded by the amount of ATP initially added through HPLC. 1 mL of PBS solution (pH 7.4 or pH 5.5) was added to the recovered Fe-ATP self-assemblies, and the mixture was then placed in a dialysis bag (SnakeSkin). TM The tube was then transferred to Dialysis Tubing (7 kDa MWCO).

[0118] The dialysis bag containing the Fe-ATP self-assembly was placed in a vial containing 4 mL of PBS solution (pH 7.4 or pH 5.5) and stored at room temperature. 40 μL of the supernatant was collected from the vial containing the dialysis bag containing the Fe-ATP self-assembly on days 1, 3, 5, and 7, and the released Fe or ATP concentration was measured and calculated. PBS solution was added to the vial to maintain a consistent total volume.

[0119] The cumulative ATP release profile was obtained using an HPLC (Arc HPLC Core System, Waters) with an analytical column (XBridge BEH C18, 130 Å, 4.6 mm × 250 mm, 5 μm particle size). Mobile phases A and B were prepared as follows: For mobile phase A, 0.06 mol / L K2HPO4, 0.04 mol / L KH2PO4, and 0.1 mol / L KOH were dissolved in deionized water (pH 7.0). Mobile phase B was a PBS solution. The elution program was as follows: A / B = 100 / 0 (v / v) for 2 min; A / B = 95 / 0 (v / v) for 2 min; A / B = 80 / 20 (v / v) for 2 min; A / B = 75 / 25 (v / v) for 1.3 min; and A / B = 100 / 0 (v / v) for 1.7 min. After running, the mobile phase A / B = 100 / 0 (v / v) was held constant for another 1 min. The flow rate was isocratic at 1.2 mL / min, the injected sample volume was 20 μL, and UV-vis absorbance was monitored at 260 nm. The cumulative Fe release profile was also obtained using an Fe analyzer (QuantiChrom) to obtain the cumulative Fe release profile at various pH levels. TM Iron Assay Kit, Bioassay systems) was used (Figure 7 and Figure 14 below).

[0120] 2.1. Confirmation of AMP release properties using Fe-AMP self-assemblies The AMP release properties were confirmed using Fe-AMP self-assembly.

[0121] Cumulative AMP release profiles were obtained to confirm sustained AMP release during AMP-induced fracture healing. To prepare Fe-AMP self-assemblies, adenosine-5'-monophosphate disodium salt (Adenosine-5'-triphosphate disodium salt, CAS: 51963-61-2, Daejung Reagents) was used instead of adenosine-5'-triphosphate disodium salt (Adenosine-5'-monophosphate disodium salt, CAS: 4578-31-8, Alfa Aesar). The Fe-ATP self-assemblies were collected in the same manner as the samples prepared to confirm Fe or ATP release characteristics.

[0122] From the vials containing the dialysis bags containing the Fe-AMP self-assemblies, 400 μL of supernatant was collected on days 1, 3, 7, 14, and 21, and the concentration of released AMP was measured by HPLC. The cumulative AMP release profile was obtained by HPLC using the same method as used in the cumulative ATP release test (see Figure 26 below).

[0123] 3.IVIS L-012 ROS signal analysis The luminescent probe L-012 (Wako Chemical, Korea) was dissolved in ultrapure water HO and prepared fresh immediately before the experiment. A 50 μl injection of 25 mg / kg was administered subcutaneously to the back of the mice.

[0124] CL (Chemiluminescence) emission evaluation is indiGO TM Using software and an IVIS (In vivo Imaging System, NightOWL II LB 983, Berthold Technologies GmbH, Germany), imaging was initiated immediately after L-012 injection. To collect sufficient CL emission data, mice were exposed for 20 seconds each time. CL emission from the mouse skin was recorded and visualized in real time, and then CL emission in the region of interest was quantified as total flux (photons / second).

[0125] Methods for evaluating cancer cell killing, osteoarthritis, and bone defect treatments using self-assemblies Experimental example 1 (cancer cell death, osteosarcoma model) To synthesize Fe-ATP self-assemblies, FeCl2 solution and ATP (adenosine triphosphate) solution were prepared using deionized water (DI water). 5 mL of 40 mM FeCl2 solution and 5 mL of 40 mM ATP (adenosine triphosphate) solution were mixed in deionized water to prepare various concentrations. 5 mL of 20 mM FeCl2 solution and 5 mL of 20 mM ATP (adenosine triphosphate) solution were added at the same concentration and mixed for 1 minute using a vortex mixer (Vortex Genie 2, Scientific Industries) to prepare a mixed solution. The mixed solution was kept at room temperature for 12 hours to induce self-assembly and produce Fe-ATP self-assemblies. The solution containing Fe-ATP self-assemblies was centrifuged at 10,000 rpm for 5 minutes. To remove unreacted reagents, the supernatant was discarded, deionized water was added, and the solution was centrifuged twice. After washing, 5 mg of the collected Fe-ATP self-assembled particles were dispersed in 1 mL of deionized water, and 20 μL of the dispersion was injected into mice.

[0126] The mice used in this experiment were 8-week-old male Balb / c nude mice (Orient, Seongnam, South Korea). To confirm the efficacy of Fe-ATP self-assembly in osteosarcoma, they were compared with PBS (phosphate-buffered saline), ATP (Daejung's Reagent, CAS: 51963-61-2), DOX (doxorubicin), Fe-ATP, Fe-ATP(mag), Fe3O4, and Fe3O4(mag), as shown in Table 2. Fe-ATP was injected at 5 mg / kg, and Fe3O4 at 7 mg / kg.

[0127] [Table 2] Before surgery, mice were anesthetized with a mixture of 50-70% medical oxygen and 2% isoflurane (CAS#26675-46-7) at 2 L / min. After an incision was made in the skin over the knee, the knee joint was identified, and the patellar muscle was exposed and then incised to expose the top of the tibia. A hole was then made in the exposed area with a syringe, and pre-prepared KHOS osteosarcoma cells (1x107 / mL, 100 µL) were injected into the bone marrow. The skin was then sutured. After the osteosarcoma cells were injected into the mice, the antibiotic enrofloxacin (Baytril, Bayer, CAS#93106-60-6) was diluted 5-fold in saline and injected once daily for 5 days after surgery to prevent infection. The analgesic ketoprofen (Ketopro inj, Unibio Co., Ltd, CAS#22071-15-4) was administered throughout the day after surgery to relieve pain.

[0128] Before injection of the Fe-ATP self-assemblies, mice were anesthetized by inhalation using a mixture of 2% isoflurane in oxygen at a rate of 2 L / min. Four weeks after surgery, the groups listed in Table 2 received six drug injections at 7-day intervals for a total of six weeks. 20 μL of Fe-ATP self-assemblies were injected into the tumor site. To compare the effects of existing chemotherapy drugs, 2 mg / kg of doxorubicin (Doxorubicin hydrochloride, AD mycin, Boryung Pharmaceutical, CAS#23214-92-8) was also injected. The Fe3O4 group received 5 mg / kg of Fe ions, injected in the same volume (20 μL). The Fe-ATP self-assemblies were injected at a dose of 5 mg / kg (self-assembly weight / mouse weight), and the Fe3O4 self-assemblies were injected at a dose of 7 mg / kg (self-assembly weight / mouse weight). As a comparison, the PBS group received the same volume of 20 μL of PBS (pH 7.4). To confirm magnetic targeting, a 270 mT circular neodymium magnet (5 mm diameter, 2 mm height) was fixed at the injection site with Tegaderm and surgical tape in the groups indicated by (mag).

[0129] After 6 weeks, the mice were euthanized using CO2, and tumor tissue samples were collected from the paws for histological examination (histology, Department of Pathology, Korea University, Seoul, Korea).

[0130] Experimental Example 2 (Osteoarthritis Model) 1 mg of Fe-ATP self-assembly particles prepared in the same manner as in Experimental Example 1 was dispersed in 10 mL of deionized water, and 20 μL of the dispersion was injected into rats.

[0131] The mice used in this experiment were 8-week-old Sprague-Dawley rats. To confirm the efficacy of Fe-ATP self-assemblies in treating osteoarthritis, they were compared with PBS (phosphate-buffered saline) (no treatment), Fe-ATP, and Fe-ATP (magnesium hydroxide) as shown in Table 3 below. The injection dose of Fe-ATP self-assemblies was 8 μg / kg (self-assembly weight / rat weight). Since the synovial fluid of one rat is approximately 100 μL, 2 μg of Fe-ATP self-assemblies were dissolved in 20 μL of PBS and injected. The ATP release profile using Fe-ATP self-assemblies (see Figure 14) showed that at room temperature, Fe-ATP self-assemblies released approximately 7% of their ATP daily and approximately 28% over 7 days. Therefore, when 8 μg / kg (weight of self-assembly / weight of rat) of Fe-ATP self-assembly is injected into a rat, the ATP concentration in the rat's synovial fluid becomes 5-8 μM, promoting M2 macrophages.

[0132] [Table 3] Experiments were performed on rats in the following order, and animals were subjected to a 7-day quarantine / acclimation process before the experiments were performed.

[0133] First, rats were intramuscularly injected with 10 mg / kg of alfaxalone (10 mg / kg, CAS#23930-19-0) and 10 mg / kg of xylazine hydrochloride (10 mg / kg, CAS#23076-35-9), and the mice were subjected to inhalation anesthesia using a mixture of oxygen and 2% isoflurane infused at 2 L / min. To prevent infection, the antibiotic enrofloxacin (Baytril, Bayer, CAS#93106-60-6) was diluted 5-fold with saline and injected at a dose of 5 mg / kg once daily for 5 days after surgery. To relieve pain, the analgesic ketoprofen (Ketopro inj, Unibio Co., Ltd, CAS#22071-15-4) was intradermally injected at a dose of 5 mg / kg for 1 day after surgery.

[0134] To perform anterior cruciate ligament transection (ACLT), the skin at the knee was incised, the knee joint was identified, and the patella tendon was laterally moved to expose the anterior cruciate ligament. The exposed anterior cruciate ligament was cut from the center to the first third using ophthalmic scissors, and a Lachman test was performed to confirm whether it was cut or not. The knee was then washed with saline, and the treatments listed in Table 3 were performed. For the groups listed in Table 3, treatments were administered by injection into the synovial fluid in the joint cavity. Each group received eight injections of drugs at seven-day intervals for a total of eight weeks. 20 μL of Fe-ATP self-assembly and 20 μL of PBS were injected in equal volumes. In the Fe-ATP(mag) group, 20 μL of Fe-ATP self-assembly was injected simultaneously. To confirm magnetic targeting of the Fe-ATP self-assembly, a 270 mT circular neodymium magnet (8 mm diameter, 2 mm height) was fixed to the joint with surgical tape. After each treatment, the skin of the knee was re-suturized, and after 8 weeks, plain radiography, micro-computed tomography (micro-CT), and histological analysis were performed.

[0135] For plain radiography (weeks 0, 2, 4, 6, and 8), 10 mg / kg of alfaxalone (CAS#23930-19-0) and 10 mg / kg of xylazine hydrochloride were administered. General anesthesia was administered intramuscularly via injection of xylazine hydrochloride (10 mg / kg, CAS#23076-35-9). Plain radiography equipment (Cios Alpha, Siemens) was used to confirm ACLT and the presence or absence of peripheral fractures immediately after surgery. Plain radiography was then performed every 2 weeks for 8 weeks to observe the radiographic progress and score the lesions according to the Kellgren-Lawrence score.

[0136] For micro-CT (8 weeks) and histological examination (8 weeks), rats were anesthetized by intramuscular injection of 10 mg / kg alfaxalone (10 mg / kg, CAS#23930-19-0) and 10 mg / kg xylazine hydrochloride (10 mg / kg, CAS#23076-35-9) and then euthanized with CO2 gas. Knee cartilage was harvested and fixed in 4% paraformaldehyde solution. A micro-CT scan (Genoss, Suwon, Korea) was performed to evaluate the state of cartilage regeneration.

[0137] Experimental example 3 (bone defect model) 3D Fe-AMP self-assemblies were fabricated in the form of micropore-containing scaffolds. For the 3D Fe-AMP self-assemblies (macroporous bone scaffolds), a PMMA (poly(methyl methacrylate)) leaching method was used. First, 300 mg of PMMA (125-150 μm diameter, Bangs Laboratories, BB05N) was filled into a cylindrical polyethylene mold (8 mm diameter). 200 μL of 2 M FeCl2 solution in deionized water was added to the mold and gently mixed using a vortex mixer for 5 minutes. Next, 200 μL of 2 M AMP solution in deionized water was added to the mold and vigorously mixed using a vortex mixer for 5 minutes. The mold containing the mixture was sealed and kept at 25°C for 1 day. 3D Fe-AMP self-assemblies were then fabricated. The Fe-AMP aggregates were separated using a mold and immersed in 50 mL of dichloromethane (DCM) for 3 days under shaking conditions (100 rpm) to leach the PMMA. The DCM was replaced every 24 hours. After 3 days, the 3D Fe-AMP aggregates, in which the Fe-AMP self-assemblies had aggregated into a 3D form, were immersed in 50 mL of deionized water for 30 minutes and then dried at room temperature. The 3D Fe-AMP aggregates were cut into 1 cm or 0.8 cm pieces and sterilized by UV irradiation for 30 minutes before use in bone repair.

[0138] The rabbits used in this experiment were New Zealand White males aged 16 to 20 weeks. To confirm the efficacy of 3D Fe-AMP aggregates in treating bone defects, they were divided into a control group (no treatment), short Fe-AMP, short Fe-AMP (mag), long Fe-AMP, and calcium phosphate (Ca-phosphate) as shown in Table 4 below. Here, short Fe-AMP was a 0.8cm Fe-AMP aggregate, long Fe-AMP was a 1cm Fe-AMP aggregate, and (mag) was a 270mT circular neodymium magnet (8mm diameter, 2mm height) fixed with surgical tape to confirm magnetic targeting.

[0139] [Table 4] Experiments were performed on rabbits in the following order, and animals were subjected to a 7-day quarantine / acclimation process before the experiments were performed.

[0140] To create the large bone defect model (0-8 weeks old), mice were given deep anesthesia (xylazine, alfalaxone, and isoflurane) prior to surgery, and were administered an antibiotic (enrofloxacin) and an analgesic (ketoprofen). Xylazine (5mg / kg IM) and alfaxalone (3mg / kg IV) were injected intramuscularly to induce anesthesia. The antibiotic enrofloxacin (5mg / kg SC) and the analgesic ketoprofen (5mg / kg IM) were then injected intradermally. After that, mice were anesthetized by inhalation using a mixture of 2% isoflurane in oxygen, infused at 2L / min.

[0141] After confirming complete anesthesia, a 7cm incision was made in the rabbit's femur, and the femur was accessed via an anterolateral approach. A 1cm bone defect was then marked in the femoral shaft, and a metal pin was used to drill the resection site. A power surgical saw (Colibri II, Depuy Synthes) was used to partially resect only the area of ​​the resection that would be covered by the metal plate. A 6-hole 2.7 locking compression plate (LCP2.7 straight (6 holes, L58mm) - Depuy Synthes) was placed in the 1.5cm bone defect to allow for fixation of three proximal and two distal metal screws. The three proximal and two distal metal screws were then firmly fixed in place, ensuring the rabbit's bone was not broken. The partially resected bone resection site was then completely resected using a power surgical saw (Colibri II, Depuy Synthes). This was done seriously because rabbit bones are very brittle and easily break.

[0142] After 3-D Fe-AMP aggregates or bone graft material (main component: calcium triphosphate, NeoBone, SN Biologics, Suwon, Republic of Korea) was inserted into the bone defect, fascia and skin sutures were applied. In the Short Fe-ATP (mag) group, to control magnetic migration for cell recruitment, a 270 mT circular neodymium magnet (20 mm diameter, 2 mm height) was placed on the surgical site, alternating between the upper and lower sides three times daily, and secured with Tegaderm and surgical tape. After 8 weeks of induction membrane formation and maturation, the animals were injected with the antibiotic enrofloxacin (5 mg / kg SC) and the analgesic ketoprofen (5 mg / kg IM) for 3 days after surgery. To evaluate the experimental results, plain radiography, micro-computed tomography (micro-CT), and histology (8 weeks) were performed.

[0143] Plain radiographs (weeks 0, 2, 4, 6, and 8) were performed using radiography equipment (Cios Alpha, Siemens) after anesthesia with an intramuscular injection of xylazine (5 mg / kg IM) and alfaxalone (3 mg / kg IV). Immediately after the primary surgery, metal plate fixation and the presence or absence of peripheral fractures were confirmed. Clinical progress was then monitored until 4 weeks of induction membrane maturation. If abnormalities were noted, plain radiographs were taken 2 weeks after the primary surgery for radiographic follow-up. Plain radiographs were then taken every 2 weeks for a total of 8 weeks, and radiographic progress was monitored and scored using the RUST score. Eight weeks after surgery, bone fixation was assessed based on fixation of at least 3 of the 4 cortical bones on two plain radiographs (anteroposterior and lateral).

[0144] Micro-computed tomography (Micro-CT) was performed on the organs (Genoss, Suwon, Korea) at 0 and 8 weeks. Eight weeks after surgery, the rabbits were sacrificed, and the femurs were obtained (KCl). They were fixed in 4% paraformaldehyde solution and then subjected to Micro-CT scans. The volume of the reconstructed bone was evaluated using 3D software (provided by Genoss).

[0145] Histological examination (8 weeks) was performed after deep anesthesia prior to euthanasia. Euthanasia was performed using potassium chloride (2 mmol / kg, IV) followed by anesthesia with twice the prescribed dose of xylazine (5 mg / kg IM) and alfaxalone (3 mg / kg IV). Potassium chloride (2 mmol / kg, IV) was then injected to induce euthanasia. Cardiac arrest was confirmed after euthanasia. Organ toxicity and peri-bone muscle tissue examination were performed using H&E (Department of Pathology, Korea University, Seoul, Korea), and bone immunohistochemistry was performed using IHC (Osteocalcin, Genoss, Suwon, Korea).

[0146] Basic performance of self-assembly and evaluation results of cancer cell killing, osteoarthritis, and bone defect treatment using self-assembly 3 to 32, the basic performance of the self-assemblies produced according to the embodiments of the present invention and the evaluation results of cancer cell killing, osteoarthritis, and bone defect treatment using the self-assemblies are shown.

[0147] Figure 3 shows an SEM image of the Fe-ATP self-assemblies and the results of measuring their paramagnetism. The paramagnetism of the Fe-ATP self-assemblies was measured using a vibrating sample magnetometry (VSM). Figure 3 shows an SEM image of the Fe-ATP self-assemblies prepared in Preparation Example 1, confirming that they were prepared in the form of uniform spherical particles with an average diameter of 600 nm.

[0148] Fe-ATP self-assembly is 2+ It was confirmed that iron ions and ATP primarily form self-aggregates through coordination bonds, and that the prepared Fe-ATP self-aggregates aggregate through hydrogen bonds or π-π interactions between adjacent Fe-ATP self-aggregates. It was also confirmed that the prepared Fe-ATP self-aggregates were spherical with a nearly uniform average diameter. Furthermore, after forming a magnetic field using a 270 mT circular neodymium magnet (8 mm diameter, 2 mm height), the Fe-ATP self-aggregates moved. In other words, it was confirmed that the Fe-ATP self-aggregates prepared in Preparation Example 1 possess reversible magnetic properties, i.e., paramagnetism, and thus move when attracted to an external magnet.

[0149] Figure 4 shows SEM images of Fe-ATP self-assemblies with average diameters of 150 nm and 70 nm, respectively, according to one embodiment of the present invention. Figure 4 shows the Fe-ATP self-assemblies prepared in Preparation Examples 2 and 3, confirming that the diameter of the self-assemblies can be controlled by the iron ion concentration and ATP concentration. Using the same preparation method, the Fe-ATP self-assemblies had an average diameter of 150 nm when the iron ion concentration was 1 mM and the ATP concentration was 1 mM, as in Preparation Example 2. The Fe-ATP self-assemblies had an average diameter of 70 nm when the iron ion concentration was 0.1 mM and the ATP concentration was 0.1 mM, as in Preparation Example 3. In other words, increasing the iron ion concentration or the ATP concentration increased the average diameter of the Fe-ATP self-assemblies.

[0150] Figure 5 is a TEM image of Fe-AMP self-aggregates. Figure 5 shows Fe-AMP self-aggregates prepared using iron ions and AMP according to Preparation Example 4. It was confirmed that the Fe-AMP self-aggregates were formed in a 3D structure with micropores, with multiple particles agglomerating together. That is, it was confirmed that the Fe-AMP self-aggregates were formed in a 3D structure with micropores between the aggregated Fe-AMP self-aggregates.

[0151] 6 to 12 show the experimental results of Experimental Example 1. FIG.

[0152] Figure 6 is a diagram showing the schematic diagram of inducing ferroptosis in cancer cells using Fe-ATP self-assemblies. Figure 6 shows experimental example 1, in which Fe-ATP self-assemblies implanted in a living body were transferred by applying a magnetic force outside the living body. Furthermore, when the Fe-ATP self-assemblies were transferred, they were transferred to the targeted cancer cells, and then the Fe-ATP self-assemblies were self-decomposed to release iron ions (Fe 2+ ) and induced ferroptosis in cancer cells. That is, the Fe-ATP self-assemblies according to this embodiment can be targeted to specific cancer cells by self-disassembly and by moving the position of the Fe-ATP self-assemblies using a magnetic field. Therefore, the Fe-ATP self-assemblies can enable the iron ion-mediated Fenton reaction for cancer treatment using ferroptosis.

[0153] FIG. 7 is a graph showing the amount of iron ions released from Fe-ATP self-assemblies at pH 5.5 and pH 7.4, as measured using an iron assay kit. 2+ Before confirming the effect of Fe-ATP, we used PBS, which is similar to biofluids, to induce Fe-ATP self-assembly. 2+ The amount of released Fe-ATP was confirmed. At both pH 5.5 and pH 7.4, all of the Fe-ATP self-assemblies were disassembled, and the iron ions, Fe 2+It was confirmed that Fe was released at pH 5.5 compared to pH 7.4. 2+ It was confirmed that Fe was released more quickly and in greater amounts. 2+ The release of Fe is accelerated in relatively acidic conditions, and by changing the ambient conditions of the Fe-ATP self-assembly, 2+ It was confirmed that the release rate and amount of Fe could be controlled. 2+ The amount of released Fe-ATP can be varied by designing the Fe-ATP self-assembly in various ways.

[0154] Figure 8 shows the intracellular ROS concentration measured by DCFDA staining under each condition. Experiments under each condition were carried out on cells before experiments on mice, as shown in Experimental Example 1 below. As shown in Table 5 below, KHOS cancer cells were used and divided into a control group treated with PBS only, DOX only, Fe-ATP treatment, ATP only treatment, and FeCl2 treatment. In the case of FeCl2, the dissolved Fe 2+ and Cl - Since it is decomposed into Fe, the Fe released by Fe-ATP 2+ The Fe-ATP self-assembly was added for comparison. It was confirmed that ROS generation was promoted when Fe-ATP self-assemblies were used compared to the control group (no treatment) in which only PBS was injected at the same volume. Furthermore, when Fe-ATP self-assemblies were used, ROS generation was promoted more than when ATP alone was used. This is attributed to the effect of iron ions generated by the self-decomposition of Fe-ATP self-assemblies.

[0155] It was confirmed that the Fe-ATP self-assembly of the present invention generates ROS more effectively than doxorubicin (DOX), which is commonly used in cancer cell therapy. Figure 8 shows cancer cells, in which the internal concentration of hydrogen peroxide is higher (10 μM-100 μM) than in normal cells (20 nM). The Fe-ATP self-assembly of the present invention is self-decomposed and released. 2+The Fenton reaction with ROS generates excessive amounts of ROS, which leads to cancer cell death through ferroptosis.

[0156] [Table 5] Figure 9 shows the results of analyzing the ROS signal in IVIS L-012 two hours after injection of the substance. Figure 9 is for Experimental Example 1, and it was confirmed that Fe-ATP self-assembly significantly stimulates ROS generation in vivo. The control group, PBS, produced almost no ROS, and Fe3O4 showed the same results as when treated with PBS alone. This is because Fe3O4 is not decomposed in vivo, and Fe 2+ This is believed to be because they do not release ROS. Furthermore, the Fe-ATP self-assemblies according to this embodiment exhibited a higher effect than doxorubicin (DOX). Doxorubicin (DOX), which is commonly used in cancer treatment, has almost no effect on ROS release in a short period of time, but it was confirmed that Fe-ATP self-assemblies generate excessive ROS even in a short period of time, making them more effective in killing cancer cells.

[0157] Figure 10 shows the sustained Fe-ATP binding of magnetically targeted Fe-ATP self-assemblies. 2+ These results confirmed the ferroptosis of cancer cells through the release of Fe-ATP. It was confirmed that Fe-ATP self-assemblies exhibit superior efficacy in cancer cell therapy compared to PBS and Fe3O4. Furthermore, when an external magnetic field, Fe-ATP (mag), was applied to the Fe-ATP self-assemblies, the Fe-ATP self-assemblies were magnetically targeted, demonstrating increased efficacy in cancer cell therapy. It was confirmed that magnetically targeted Fe-ATP self-assemblies suppressed cancer development to a level equivalent to that of doxorubicin (DOX), a commonly used anticancer drug. On the other hand, when Fe3O4 alone was used, it was confirmed that the in vivo degradation efficiency was low, regardless of whether it was magnetically targeted, and thus it was unable to suppress cancer development.

[0158] Figure 11 shows that magnetically targeted Fe-ATP self-assemblies effectively treat osteosarcoma cancer, similar to doxorubicin. In Figure 11, a large number of nuclei, appearing purple in H&E staining, indicates the presence of many cancer cells, while a large number of DNA fragments, appearing brown in TUNEL staining, indicates the death of many cancer cells. Furthermore, the cancer tissue was shown by H&E staining and TUNEL staining using hematoxylin and eosin. Immunohistochemistry (IHC) staining revealed that the released LPO, GPX4, and Xc receptors due to cancer cell death by Fe-ATP self-assemblies were linked to the release of Fe-ATP self-assemblies. 2+ It was confirmed that α-glucan contributes to ferroptosis stimulated by the Fenton reaction.

[0159] Figure 12 shows the results of examining the toxicity of Fe-ATP self-assemblies after transplantation into a living body. In Figure 12, even after transplantation of Fe-ATP self-assemblies under a magnetic field in an osteosarcoma mouse model, the control group showed the same results as PBS, confirming that no toxicity was observed in the major organs of the living body. That is, the Fe-ATP self-assemblies according to this embodiment self-decompose, resulting in the formation of Fe 2+ It was confirmed that even when released, it is not toxic to the human body, and is also not toxic when a magnetic field is applied.

[0160] 13 to 22 show the experimental results of Experimental Example 2. FIG.

[0161] Figure 13 shows the results of confirming the chondroprotective effect of magnetically targeted Fe-ATP self-assemblies. Figure 13, which is for Experimental Example 2, shows that magnetically targeted Fe-ATP self-assemblies self-disassemble to release ATP, which induces anti-inflammatory macrophage polarization and is effective in treating osteoarthritis.

[0162] Figure 14 is a graph showing the cumulative ATP release profile from Fe-ATP self-assemblies measured by HPLC. Before examining the effects of ATP released from Fe-ATP self-assemblies in Experimental Example 2, the amount of ATP released from Fe-ATP self-assemblies was examined using PBS, a similar biological fluid. It was confirmed that the Fe-ATP self-assemblies according to this embodiment self-decompose, releasing iron ions and ATP, as discussed above. It was also confirmed that the Fe-ATP self-assemblies released 30% of their ATP over approximately 25 days. The amount of ATP released from the Fe-ATP self-assemblies according to this embodiment can be varied by designing the Fe-ATP self-assemblies in various ways.

[0163] 15 to 17, experiments were performed on cells under various conditions before experiments on rats in Experimental Example 2 below. As shown in Table 6 below, the conditions were divided into a control group treated with PBS only, Fe-ATP treatment, ATP only treatment, FeCl2 treatment, and Fe3O4 treatment.

[0164] [Table 6] Figure 15 shows the results of confirming the disassembly of Fe-ATP self-aggregates. Referring to Figure 15, immunofluorescent staining of Arg-1 (an M2 polarization marker), iNOS (an M1 polarization marker), and Dapi was performed under each condition. PBS (no treatment), FeCl2, and Fe3O4 did not show any Arg-1 (an M2 polarization marker), whereas the ATP-containing samples, Fe-ATP self-aggregates, and ATP alone, showed strong M2 polarization marker Arg-1. It was confirmed that Fe-ATP self-aggregates disassembled to release ATP, which enables ATP-mediated anti-inflammatory polarization of macrophages for the treatment of osteoarthritis.

[0165] Figure 16 shows the results of confirming macrophage polarization due to the disassembly of Fe-ATP self-assemblies. Referring to Figure 16, it was confirmed that Fe-ATP self-assemblies self-disassemble to release ATP, enabling ATP-mediated anti-inflammatory polarization of macrophages for the treatment of osteoarthritis. This was confirmed by flow cytometry.

[0166] Figure 17 shows the results of Western blotting to confirm the disassembly of Fe-ATP self-aggregates. We confirmed that Fe-ATP self-aggregates disassembled, enabling AIP-mediated anti-inflammatory polarization of macrophages. We also confirmed that low concentrations of ATP stimulated P2Y1 signaling, which promotes M2 polarization of macrophages, and inhibited P2X7 signaling, which suppresses M2 polarization of macrophages.

[0167] Figure 18 shows C-Arm and 3-D microCT images of the knee joint to confirm the therapeutic effect of Fe-ATP self-assembly on osteoarthritis. C-Arm images of the knee joint were obtained at 0, 2, 4, 6, and 8 weeks to identify ACLT and peripheral fractures in the osteoarthritis model. 3-D microCT images were obtained at 8 weeks to confirm ACLT and peripheral fractures in the osteoarthritis model. Compared to PBS (no treatment), Fe-ATP self-assemblies and magnetically targeted Fe-ATP self-assemblies were confirmed to be effective in treating osteoarthritis.

[0168] Figure 19 shows 2-D micro-CT images, H&E stained images, and safranin-o (SO) stained images of the knee joint of an osteoarthritis model at 8 weeks. Figure 19 shows ACLT and peripheral fractures in the cases treated with Fe-ATP self-assemblies according to the present embodiment. It was confirmed that Fe-ATP self-assemblies and magnetically targeted Fe-ATP self-assemblies protect the cartilage in the knee joint and prevent cartilage damage.

[0169] Figure 20 shows H&E, safranin-O (SO)-stained, and collagen X-stained images of the knee joint of an 8-week osteoarthritis model. ACLT and peripheral fractures were confirmed in this 8-week osteoarthritis model. Fe-ATP self-assemblies and magnetically targeted Fe-ATP self-assemblies alleviated inflammation through anti-inflammatory macrophage polarization and suppressed the formation of hypertrophic differentiated chondrocytes, which mediate cartilage-to-bone metastasis. In particular, magnetically targeted Fe-ATP self-assemblies demonstrated even greater efficacy than Fe-ATP self-assemblies. Collagen X stained hypertrophic differentiated chondrocytes, which were found to promote cartilage-to-bone metastasis and exacerbate osteoarthritis.

[0170] Figure 21 shows images of iNOS and Arg-1 staining in the synovium of the knee joint of an osteoarthritis model at 8 weeks. Figure 21 shows the ACLT and peripheral fractures of the osteoarthritis model. It was confirmed that Fe-ATP self-assemblies promote M2 polarization of macrophages through P2Y1 signaling, protecting the knee joint from cartilage degradation, and that magnetically targeted Fe-ATP self-assemblies were particularly effective.

[0171] Figure 22 shows the toxicity of Fe-ATP self-assemblies observed in a rat model of osteoarthritis. Fe-ATP self-assemblies were implanted into various locations under a magnetic field, and no in vivo toxicity was observed in major organs. That is, it was confirmed that the Fe-ATP self-assemblies according to this embodiment are not toxic to the human body even when they self-decompose and release ATP, and are also not toxic when a magnetic field is applied.

[0172] 23 to 32 show the experimental results of Experimental Example 3. FIG.

[0173] FIG. 23 is a diagram schematically illustrating the bone regeneration effect of 3D Fe-AMP self-assembly.

[0174] Figure 23 shows experimental example 3, demonstrating that 3D Fe-AMP self-assemblies exhibit bone regeneration effects through osteoblast differentiation of stem cells mediated by adenosine A2B receptor signaling. 3D Fe-AMP self-assemblies were formed into 3D macroporous aggregates that enabled magnetic motility-based cell recruitment for degraded-AMP-mediated fracture healing through adenosine A2B receptor signaling.

[0175] 24 is an SEM image showing a micropore structure formed by aggregation of 3D Fe-AMP self-assemblies. The Fe-AMP self-assemblies according to this embodiment can be manufactured in a 3D form having a micropore structure, and their size can be controlled in various ways, from several micrometers to several tens of centimeters. In addition, the manufactured Fe-AMP self-assemblies can be self-degraded in vitro or in vivo, and when self-degraded, they release iron ions, Fe 2+ It was confirmed that the enzyme released AMP.

[0176] Figure 25 shows the magnetic targeting of 3D Fe-AMP self-assemblies. The application of an external magnetic field to the 3D Fe-AMP self-assemblies also allowed for the control of their movement.

[0177] 26 is a graph showing AMP released over time from Fe-AMP self-assemblies. Prior to examining the effects of AMP released from Fe-AMP self-assemblies in Experimental Example 3, the amount of AMP released from Fe-AMP self-assemblies was examined using PBS, a similar fluid to biological fluids. It was confirmed that the Fe-AMP self-assemblies according to this embodiment self-decompose over time to release AMP, with the release lasting for approximately 25 days and approximately 40% released. The amount of AMP released from the Fe-AMP self-assemblies according to this embodiment can be varied by variously designing the Fe-AMP self-assemblies.

[0178] 27 and 28, experiments were carried out on cells under various conditions before conducting experiments on rabbits in Experimental Example 3 below. The conditions were classified as shown in Table 7 below, into a control group treated with PBS only, Fe-AMP treatment, AMP treatment only, adenosine and PO4 (adenosine + PO4) combined treatment, adenosine treatment, PO4 treatment, FeCl2 treatment, and osteogenic induction medium treatment. In the case of FeCl2, the dissolved Fe 2+ and Cl - Since it is decomposed into Fe, the Fe released by Fe-ATP 2+ Added for comparison.

[0179] [Table 7] Figure 27 shows the results confirming that AMP-degraded adenosine released from 3D Fe-AMP self-assemblies promotes osteogenic differentiation. The AMP-degraded adenosine released from Fe-AMP self-assemblies was analyzed by immunofluorescent staining for RUNX2 and osteocalcin, along with actin and DAPI-positive nuclei and ALP chemical staining, similar to osteogenic differentiation induction medium. Fe-AMP self-assemblies induced osteogenic differentiation of stem cells in response to osteogenic induction medium, which promotes osteogenic differentiation, and adenosine also induced osteogenic differentiation. In other words, Fe-AMP self-assemblies induce osteogenic differentiation of stem cells through adenosine signaling, which was confirmed by immunofluorescence staining of RUNX2 and osteocalcin, as well as actin and Dapi-positive nuclei and ALP chemical staining.

[0180] Figure 28 shows the results of Western blotting, confirming osteogenic differentiation through CD73-mediated AMP-degrading adenosine released from 3D Fe-AMP self-assemblies. As shown in Figure 28, CD73-mediated AMP-degrading adenosine released from Fe-AMP self-assemblies promotes osteogenic differentiation through adenosine A2B receptor signaling. Addition of an A2B receptor inhibitor (PSB 603) inhibited adenosine A2B receptor signaling, thereby suppressing osteogenic differentiation of hMSCs. We confirmed that CD73 degrades AMP to adenosine and PO4, promoting osteogenic differentiation through adenosine A2B receptor signaling.

[0181] Figure 29 shows C-Arm images of femurs at 0, 2, 4, 6, and 8 weeks to confirm fracture. Referring to Figure 29, it was confirmed that the 3D Fe-AMP self-assemblies of the present invention exhibited similar effects to calcium phosphate, a commonly used implant for fracture treatment, compared to the control group. Specifically, while Short Fe-AMP self-assemblies were more effective in fracture treatment than the control group, magnetically targeted Short Fe-AMP(mag) self-assemblies were even more effective in fracture treatment than Short Fe-AMP self-assemblies. It was also confirmed that Short Fe-AMP(mag) self-assemblies and Long Fe-AMP self-assemblies heal fractures in a manner similar to calcium phosphate. That is, when comparing Short Fe-AMP self-assemblies with Short Fe-AMP(mag) self-assemblies, it was confirmed that Short Fe-AMP(mag) self-assemblies can be imparted with a magnet, making them more effective in fracture treatment.

[0182] Figure 30 shows micro-CT images of femurs at 8 weeks to identify fractures. In Figure 30, Short Fe-AMP(mag) self-assemblies demonstrated overall fracture healing, which was attributed to cell recruitment based on magnetic migration and reduced AMP-mediated adenosine A2B receptor signaling. Long Fe-AMP self-assemblies demonstrated overall fracture healing, which was confirmed to be due to reduced AMP-mediated adenosine A2B receptor signaling. The fracture healing effects are shown in the order of Long Fe-AMP self-assemblies, Short Fe-AMP(mag) self-assemblies, and Short Fe-AMP self-assemblies, with Long Fe-AMP self-assemblies demonstrating effects similar to those of calcium phosphate. Also, in Figure 30, a lower P value indicates a greater difference between the two values. Control vs. Short Fe-AMP, p=0.074, Control vs. Short Fe-AMP (mg), p<0.001, Control p<0.001 for Long Fe-AMP, p<0.001 for Control vs. Calcium phosphate, and p=0.008 for Short Fe-AMP vs. Short Fe-AMP (mg).

[0183] Figure 31 shows H&E, osteocalcin, and TRAP staining images of fractures. Short Fe-AMP (mag) self-assemblies demonstrated overall fracture healing, which was attributed to magnetic migration-based cell recruitment and reduced AMP-mediated adenosine A2B receptor signaling. Long Fe-AMP self-assemblies demonstrated overall fracture healing, which was attributed to reduced AMP-mediated adenosine A2B receptor signaling.

[0184] Referring to Figure 31, the tissue pattern was confirmed through the H&E staining image, the degree of bone formation differentiation through the osteocalcin staining image, and the presence or absence of osteoclasts through the TRAP staining image. It was confirmed that bone was generated by connecting the middle of the defects, and considering that osteoclasts were activated, it appears that the decomposition and regeneration of the bone environment was promoted, creating an environment for bone growth.In the case of short Fe-AMP self-assemblies, it was confirmed that the newly generated bone was unable to connect all of the binding sites because a magnetic field was not applied.

[0185] Figure 32 shows the results of examining the toxicity of 3D Fe-AMP self-assemblies in vivo. After implanting the 3D Fe-AMP self-assemblies into a rabbit fracture healing model, they were examined under a magnetic field and found to have no toxicity in major organs.

[0186] As described above, the Fe-ATP self-assemblies or Fe-AMP self-assemblies according to the present embodiment are non-toxic in vivo and can self-decompose in vivo or in vitro to release iron ions or ATP / ADP, with the amount and rate of release being controllable. Furthermore, because they are paramagnetic, they can be magnetically targeted by applying an external magnetic field, controlling their movement to a specific location. Furthermore, no toxicity was observed in vivo when the Fe-ATP self-assemblies or Fe-AMP self-assemblies were magnetically targeted.

[0187] Specifically, Fe-ATP self-assemblies are effective in cancer treatment by inducing cancer cell death through ferroptosis, and Fe-ATP self-assemblies can treat osteoarthritis by releasing ATP, while Fe-AMP self-assemblies can act as a graft material for bone defects.

[0188] Those skilled in the art will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential features thereof. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. The scope of the present invention is defined by the claims set forth below rather than the above detailed description, and all modifications and variations that fall within the meaning and scope of the claims and their equivalents should be construed as being within the scope of the present invention.

Claims

1. A therapeutic agent comprising a therapeutic magnetic biomolecule-metal ion self-assembly complex, wherein the metal ion comprises an iron ion and the biomolecule comprises one or more ligands; The ligand and the iron ion are reversibly self-assembled or self-disassembled, the ligand and the iron ion are self-assembled by the first bond to form a self-assembly; the self-assembly is self-assembled by one or more of the metal ions and the ligands, A therapeutic magnetic biomolecule-metal ion self-assembly complex, wherein a plurality of self-assemblies are provided, and adjacent self-assemblies are self-bonded to each other by a second bond, wherein: The therapeutic agent comprises a therapeutic magnetic biomolecule-metal ion self-assembly complex, wherein the ligand comprises at least one of AMP (adenosine monophosphate) and ATP (adenosine triphosphate).

2. the first bond comprises a coordinate bond; The therapeutic agent comprising the therapeutic magnetic biomolecule-metal ion self-assembly complex according to claim 1, wherein the second bond comprises at least one of a hydrogen bond and a π-π interaction.

3. The self-assemblies or adjacent self-assemblies are self-assembled for a first time or self-disassembled for a second time under physiologically relevant conditions; the first time period is between 1 minute and 24 hours; 2. The therapeutic agent comprising a therapeutic magnetic biomolecule-metal ion self-assembly complex according to claim 1, wherein the second time period is from 1 day to 90 days.

4. 2. The therapeutic agent comprising the therapeutic magnetic biomolecule-metal ion self-assembly complex of claim 1, wherein the ligand comprises ATP (adenosine triphosphate), and the therapeutic magnetic biomolecule-metal ion self-assembly complex is provided in the form of individual spheres.

5. A therapeutic agent comprising a therapeutic magnetic biomolecule-metal ion self-assembly complex according to claim 1, wherein the ligand comprises AMP, and the therapeutic magnetic biomolecule-metal ion self-assembly complex is provided by aggregating a plurality of the self-assemblies into a three-dimensional aggregate having micropores.

6. The self-assembly has paramagnetic properties, 2. A therapeutic agent comprising a therapeutic magnetic biomolecule-metal ion self-assembly complex according to claim 1, wherein the movement of the self-assembly is controlled by application of an external magnetic field.

7. The self-assembly is accelerated in self-decomposition under at least one of a condition containing a chelating agent, a strong acid condition, and a strong base condition; The chelating agent may be at least one of ethylenediaminetetraacetic acid (EDTA), bipyridyl, and ferrozine; The strong acid condition has a pH of 2 to 5, 2. The therapeutic agent comprising a therapeutic magnetic biomolecule-metal ion self-assembly complex according to claim 1, wherein the strongly basic condition has a pH of 9 to 12.

8. The self-assembly generates reactive oxygen species (ROS) under conditions containing hydrogen peroxide, The therapeutic agent according to claim 1, wherein the reactive oxygen species oxidize cellular phospholipids and inhibit GPX4 (glutathione peroxidase 4) or System xc-cystine / glutamate antiporter (Xc), thereby inducing cancer cell death through ferroptosis.

9. The self-assembly has paramagnetic properties and its movement is controlled by application of an external magnetic field; 9. The therapeutic agent comprising the therapeutic magnetic biomolecule-metal ion self-assembly complex according to claim 8, wherein the self-assembly moves to target cancer cells upon application of the external magnetic field and induces the death of the cancer cells through ferroptosis.

10. 9. The therapeutic agent comprising the therapeutic magnetic biomolecule-metal ion self-assembly complex according to claim 8, wherein the self-assembly induces the death of cancer cells through ferroptosis while not exhibiting toxicity to normal cells.

11. The self-assembly is used in cancer treatment; 2. The therapeutic agent comprising the therapeutic magnetic biomolecule-metal ion self-assembly complex according to claim 1, wherein the cancer is at least one selected from the group consisting of breast cancer, colorectal cancer, rectal cancer, lung cancer, colon cancer, thyroid cancer, oral cancer, pharyngeal cancer, laryngeal cancer, cervical cancer, brain cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, pancreatic and intestinal cancer, prostate cancer, skin cancer, tongue cancer, uterine cancer, stomach cancer, bone cancer, and blood cancer.

12. the ligand comprises ATP; 2. The therapeutic agent comprising the therapeutic magnetic biomolecule-metal ion self-assembly complex according to claim 1, wherein the self-assembly releases the ATP during self-decomposition to promote M2 polarization of macrophages through the P2Y1 receptor.

13. The self-assembly promotes M2 polarization of macrophages distributed in the synovial membrane of bone joints; 13. The therapeutic agent comprising the therapeutic magnetic biomolecule-metal ion self-assembly complex according to claim 12, wherein the M2 polarization of macrophages has anti-inflammatory activity in synovial fluid of bone joints.

14. The therapeutic agent comprising the therapeutic magnetic biomolecule-metal ion self-assembly complex according to claim 13, wherein the self-assembly maintains an anti-inflammatory environment in synovial fluid of bone joints, protects bone and cartilage, and prevents, improves, or treats osteoarthritis.

15. The self-assembly has paramagnetic properties and its movement is controlled by application of an external magnetic field; The therapeutic agent comprising the therapeutic magnetic biomolecule-metal ion self-assembly complex of claim 13, wherein the self-assembly migrates to the target bone joint site upon application of the external magnetic field, protects the bone and cartilage, and prevents, improves, or treats osteoarthritis.

16. The therapeutic agent comprising the therapeutic magnetic biomolecule-metal ion self-assembly complex according to claim 13, wherein the M2 polarization of macrophages has anti-inflammatory activity in synovial fluid of bone joints, while not exhibiting toxicity to normal cells.

17. the ligand comprises AMP (adenosine monophosphate); the therapeutic magnetic biomolecule-metal ion self-assembly complex is provided by aggregating a plurality of the self-assemblies into a three-dimensional aggregate having micropores; 2. The therapeutic agent comprising the therapeutic magnetic biomolecule-metal ion self-assembly complex of claim 1, wherein the three-dimensional aggregates have an average diameter of 500 μm to 10 cm.

18. The therapeutic agent comprising the therapeutic magnetic biomolecule-metal ion self-assembly complex according to claim 17, wherein the self-assembly is used as a bone graft material to repair missing bone tissue by being transplanted into a bone defect site in the form of the three-dimensional aggregate.

19. The self-assembly has paramagnetic properties and its movement is controlled by application of an external magnetic field; The self-assembly is implanted in the form of the three-dimensional aggregate into a bone defect site; 18. The therapeutic agent comprising the therapeutic magnetic biomolecule-metal ion self-assembly complex according to claim 17, wherein the self-assembly moves in at least one direction by application of the external magnetic field.

20. The self-assembly is implanted into a bone defect site, 18. A therapeutic agent comprising a therapeutic magnetic biomolecule-metal ion self-assembly complex according to claim 17, wherein host cells are attached to the surface of the self-assembly to form a scaffold.

21. The self-assembly has paramagnetic properties and its movement is controlled by application of an external magnetic field; 21. A therapeutic agent comprising a therapeutic magnetic biomolecule-metal ion self-assembly complex according to claim 20, wherein host cells attached to the surface of the self-assembly also migrate together with the application of the external magnetic field.

22. The self-assembly is implanted into a bone defect site, 18. A therapeutic agent comprising a therapeutic magnetic biomolecule-metal ion self-assembly complex according to claim 17, wherein the self-assembly self-decomposes to release the AMP for 1 to 70 days.

23. The AMP released by the self-assembly is decomposed into adenosine on the surface of the host cell, promoting signaling of adenosine receptors on the surface of the host cell; 23. A therapeutic agent comprising a therapeutic magnetic biomolecule-metal ion self-assembly complex according to claim 22, wherein the adenosine receptor comprises an adenosine A2B receptor.

24. The AMP released from the self-assembly promotes osteogenic differentiation of mesenchymal stem cells (MSCs) by signaling the adenosine receptors of the MSCs.

23. The therapeutic agent comprising a therapeutic magnetic biomolecule-metal ion self-assembly complex according to claim 22, wherein the osteogenic differentiation of the mesenchymal stem cells (MSCs) promotes bone formation.

25. The therapeutic agent comprising the therapeutic magnetic biomolecule-metal ion self-assembly complex according to claim 18, wherein the self-assembly does not exhibit toxicity to normal cells while being transplanted into a bone defect site to regenerate the lost bone tissue.

26. The ligand includes at least one of AMP (adenosine monophosphate) and ATP (adenosine triphosphate); The self-assembly has paramagnetic properties and its movement is controlled by an externally applied magnetic field; When the ligand is one or more of AMP and ATP, the self-assembly induces cancer cell death through ferroptosis; the ligand comprises ATP and the self-assembly prevents, ameliorates, or treats osteoarthritis; or 2. The therapeutic agent comprising the therapeutic magnetic biomolecule-metal ion self-assembly complex according to claim 1, wherein the ligand comprises AMP, and the self-assembly is transplanted into a bone defect site to regenerate the lost bone tissue.

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