Magnetic biomolecular-metal ion self-assembly complex for treatment

Abstract

The present invention relates to a therapeutic magnetic biomolecular-metal ion self-assembly complex comprising: an iron ion; and one or more ligands; wherein the ligand and the iron ion reversibly self-assemble or self-decompose, and the ligand and the iron ion self-assemble by a first bond to form a self-assembly body, wherein the self-assembly body is self-assembled by one or more of the metal ion and the ligand, and wherein the self-assembly body is provided in a plurality, and adjacent self-assemblies are self-bonded by a second bond. According to the present invention, it can be effectively used for cancer treatment, osteoarthritis treatment, and bone defect treatment.

Description

Technology Field

[0001] The present invention relates to a therapeutic magnetic biomolecular-metal ion self-assembly complex, and more specifically, to a therapeutic magnetic biomolecular-metal ion self-assembly complex having an enhanced effect in the treatment of cancer, osteoarthritis, and bone defects. Background Technology

[0002] Recently, anticancer treatment relies on chemically synthesized drugs. Chemotherapy drugs, including doxorubicin, which are commonly used in cancer treatment, treat cancer by inducing apoptosis in cancer cells. On the other hand, representative anticancer drugs such as doxorubicin, cisplatin, and methotrexate are known to cause side effects on the heart, kidneys, and nerves, respectively, and these side effects present limitations in that it is difficult to prescribe them appropriately depending on the patient's condition. In particular, doxorubicin, which is widely used, has a very narrow therapeutic index (TI), and it is known that using concentrations higher than this TI causes fatal side effects.

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

[0004] Therefore, existing anticancer drugs have evolved by targeting cancer and inducing apoptosis, and while many technologies have been developed, various new methods are being researched to eliminate cancers resistant to apoptosis induced by existing drugs while maintaining the efficacy of eliminating these cancer cells and causing fewer side effects.

[0005] Arthritis is one of the most common chronic diseases in the world, but because the pathophysiology of osteoarthritis has not been fully studied, there is currently no complete cure. Osteoarthritis damages joint cartilage and causes severe pain and disability in patients, yet physiological regeneration of joint cartilage occurs only to a limited extent. Consequently, various researchers are studying the pathophysiology of osteoarthritis using animal models and seeking treatments, and recently, methods using drugs to treat osteoarthritis have been implemented. However, the drugs used to treat arthritis—such as hyaluronic acid, nonsteroidal anti-inflammatory drugs (NSAIDs), and the steroidal TAA (triamcinolone)—are limited to inhibiting inflammatory responses, and drugs that regenerate cartilage are virtually non-existent.

[0006] Furthermore, various types of cells, including mesenchymal stem cells (MSCs), chondrocytes, macrophages, and fibroblasts, exist in the joints; however, osteoarthritis worsens when the cytokine balance of the synovial fluid within the joint is disrupted, causing pro-inflammatory cytokines to increase excessively compared to anti-inflammatory cytokines.

[0007] Therefore, research is needed on drugs and treatment methods that are effective for treating bone and joint problems and can regulate cytokine balance within the joints to normal bone and joint levels.

[0008] Extensive bone defects in fractures of long bones, including the femur, are clinically very difficult to treat. Recently, the induced membrane technique has been used as a treatment method for such extensive bone defects. The induced membrane technique involves removing all necrotic or infected bone in a primary surgery, filling the defect with bone cement such as calcium phosphate (Ca-Phosphate), waiting approximately 4 to 6 weeks for the formation of an induced membrane resulting from the reaction with the bone cement, and then performing a secondary surgery to remove the bone cement and graft a large amount of autologous bone into the defect site. The BMPs used in this induced membrane technique are very expensive for actual treatment, and there are clinical challenges that need to be overcome. Furthermore, the procedure is currently performed on a limited basis using autologous bone grafts containing BMPs; there are cases where there is a shortage of autologous bone to harvest, or where the amount is insufficient even after harvesting all available autologous bone, including pelvic bone and tibia, due to the bone defect being too extensive.

[0009] Such autologous bone grafting causes many complications, such as delayed rehabilitation due to pain at the harvest site, bleeding, infection, and iatrogenic fractures, so there is a need for research on graft materials that can replace autologous bone in the induced membrane method.

[0010] (Patent Document 1) KR 10-0901127 B1 The problem to be solved

[0011] The objective of the present invention is to provide a therapeutic magnetic biomolecular-metal ion self-assembly complex that can self-assemble into various forms based on in vivo materials and does not cause side effects in vivo.

[0012] In addition, another objective of the present invention is to provide a therapeutic magnetic biomolecular-metal ion self-assembly complex that is non-toxic to living organisms, self-assembles into various forms depending on the type of ligand, and allows for easy positional control of the therapeutic magnetic biomolecular-metal ion self-assembly complex present in the body from outside the body, thereby enabling effective treatment of a targeted site. means of solving the problem

[0013] According to one aspect of the present invention, embodiments of the present invention include a therapeutic magnetic biomolecular-metal ion self-assembly complex.

[0014] In one embodiment, the therapeutic magnetic biomolecular-metal ion self-assembly complex comprises an iron ion; and one or more ligands; and the ligand and the iron ion are reversibly self-assembled or self-decomposed, and the ligand and the iron ion can self-assemble by a first bond to form a self-assembled body.

[0015] In one embodiment, the self-assembly is performed by one or more of the metal ion and the ligand, and the self-assembly may be provided in multiple numbers, with adjacent self-assemblies being self-coupled by a second bond.

[0016] In one embodiment, the first bond includes 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-assemblies or adjacent self-assemblies are self-assembled for a first time or self-decomposed for a second time under physiologically relevant conditions, 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 phosphate and 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, AEP (2-aminoethylphosphonic acid), TNA (Threose nucleic acid), GNA (glycol nucleic acid), HNA (1,5-anhydrohexitol nucleic acid), ANA (1,5-anhydroatritol nucleic acid), FANA (2'-deoxy-2'-fluoroarabino nucleic acid) and CeNA (cyclohexenyl nucleic acid).

[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 biomolecular-metal ion self-assembly complex is provided as individual spheres, and when the ligand is AMP (adenosine monophosphate), the therapeutic magnetic biomolecular-metal ion self-assembly complex may be provided as a three-dimensional aggregate having micropores formed by the aggregation of a plurality of self-assemblies.

[0022] In one embodiment, the self-assembly has paramagnetism, and the movement of the self-assembly can be controlled by the application of an external magnetic field.

[0023] In one embodiment, the self-assembly is promoted to self-decompose under at least one of conditions including a chelating agent, strong acid conditions, and strong base conditions, and

[0024] Under conditions including the above chelating agent, the chelating agent may be one or more of EDTA (ethylenediaminetetraacetic acid), bipyridyl, and ferrozine, 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 an excess of reactive oxygen species (ROS) under conditions containing hydrogen peroxide, and the reactive oxygen species oxidize the phospholipids of the cells, while inhibiting GPX4 (glutathione peroxidase 4) or System xc-cystine / glutamate antiporter (Xc), thereby inducing the death of cancer cells through ferrotosis.

[0026] In one embodiment, the self-assembly has paramagnetism and its movement is controlled by the application of an external magnetic field, and by the application of the external magnetic field, the self-assembly moves to a target cancer cell and can induce the death of the cancer cell through ferrotosis.

[0027] In one embodiment, the self-assembly may not exhibit toxicity to normal cells while inducing apoptosis of cancer cells through ferrotosis.

[0028] In one embodiment, the self-assembly is used for cancer treatment, and the cancer may be 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 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 can promote M2 polarization of macrophages through P2Y1 receptors by releasing the ATP during self-decomposition.

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

[0031] In one embodiment, the self-assembly can prevent, improve, or treat osteoarthritis by maintaining an anti-inflammatory environment of the synovial fluid of the bone joint to protect the osteochondral.

[0032] In one embodiment, the self-assembly has paramagnetism and its movement is controlled by the application of an external magnetic field, and by the application of the external magnetic field, the self-assembly moves to a target bone joint area to protect the bone cartilage, thereby preventing, improving, or treating osteoarthritis.

[0033] In one embodiment, the self-assembly promotes M2 polarization of macrophages distributed in the synovial membrane of a bone joint, and the M2 polarization of the macrophages may not exhibit toxicity to normal cells while having anti-inflammatory activity of the synovial fluid of the bone joint.

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

[0035] In one embodiment, the self-assembly can be used as a bone graft material to restore the bone tissue by implanting it into a bone defect site in the form of the three-dimensional aggregate.

[0036] In one embodiment, the self-assembly has paramagnetism and its movement is controlled by the application of an external magnetic field, and the self-assembly is implanted into a bone defect site in the form of a three-dimensional aggregate, and the self-assembly can move in one or more directions, including one direction and the other, by the application of the external magnetic field.

[0037] In one embodiment, the self-assembly is implanted into a bone defect site, and host cells can attach to the surface of the self-assembly to form a skeleton.

[0038] In one embodiment, the self-assembly has paramagnetism and its movement is controlled by the application of an external magnetic field, and a host cell attached to the surface of the self-assembly can also move together by the application of the external magnetic field.

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

[0040] In one embodiment, the AMP released from the self-assembly is broken down into adenosine on the surface of the host cell to promote signaling of the adenosine receptor on the surface of the host cell, and the adenosine receptor may include an adenosine A2B receptor.

[0041] In one embodiment, the AMP released from the self-assembler promotes osteogenic differentiation of mesenchymal stem cells (MSCs) through signaling of adenosine receptors of mesenchymal stem cells (MSCs), and the osteogenic differentiation of mesenchymal stem cells (MSCs) can promote bone formation.

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

[0043] In one embodiment, the ligand comprises one or more of AMP (adenosine monophosphate) and ATP (adenosine triphosphate), and the self-assembly has paramagnetism and is controlled to move by an externally applied magnetic field. When the ligand is one or more of AMP and ATP, the self-assembly induces the death of cancer cells through ferrotosis. When the ligand is ATP, the self-assembly prevents, improves, or treats osteoarthritis. When the ligand is AMP, the self-assembly can be implanted into a bone defect site to regenerate the defective bone tissue. Effects of the invention

[0044] According to the present invention as described above, a therapeutic magnetic biomolecular-metal ion self-assembly complex can be provided that is effective for cancer treatment by enabling targeted therapy to a local site, having no side effects, and easily inducing apoptosis.

[0045] In addition, according to the present invention, a therapeutic magnetic biomolecular-metal ion self-assembly complex can be provided to regulate the cytokine balance within a bone joint affected by osteoarthritis to the level of a normal bone joint in order to prevent the exacerbation of osteoarthritis.

[0046] In addition, according to the present invention, a therapeutic magnetic biomolecular-metal ion self-assembly complex can be provided that effectively treats bone defects and has no side effects. Brief explanation of the drawing

[0047] FIG. 1 is a schematic diagram showing the formation of a self-assembly by iron ions and ATP according to one embodiment of the present invention. FIG. 2 is a schematic diagram showing the formation of a self-assembly by iron ions and AMPs according to one embodiment of the present invention. Figure 3 shows an SEM image of the Fe-ATP self-assembled structure and the results of measuring paramagnetism. Figure 4 is an SEM image 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 is a TEM image of the Fe-AMP self-assembly. Figure 6 is a schematic diagram showing the induction of ferroptosis in cancer cells using Fe-ATP self-assemblies. Figure 7 is a graph showing the amount of iron ions released by Fe-ATP self-assemblies at pH 5.5 or pH 7.4, measured using an iron assay kit. Figure 8 shows the intracellular ROS concentration measured by DCFDA staining according to each condition. Figure 9 shows the results of analyzing the IVIS L-012 ROS signal. Fig. 10 shows the continuous Fe of a magnetically targeted Fe-ATP self-assembly. 2+ This is the result confirming the ferrotosis of cancer cells through release. Figure 11 showed that magnetically targeted Fe-ATP self-assemblies effectively treat osteosarcoma cancer similar to doxorubicin. Figure 12 shows the results of confirming the toxicity after transplanting the Fe-ATP self-assembly into the body. Figure 13 shows the results confirming the chondroprotective effect of magnetically targeted Fe-ATP self-assemblies. Figure 14 is a graph showing the cumulative ATP release profile from Fe-ATP self-assemblies measured by HPLC. Figure 15 is the result of confirming the polarization of macrophages by the degradation of Fe-ATP self-assemblies using immunofluorescence imaging. Figure 16 shows the results of confirming the polarization of macrophages by the breakdown of Fe-ATP self-assemblies through flow cytometry. Figure 17 shows the results of confirming the degradation of Fe-ATP self-assemblies by western blotting. Figure 18 shows 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 shows 2-D micro CT images, H&E staining, and safranin-o(SO) staining images of a knee joint of an osteoarthritis model at week 8. Figure 20 shows H&E staining, safranin-o(SO) staining, and collagen X staining images of an osteoarthritis model at week 8. Figure 21 is an iNOS and Arg-1 stained image of the synovium of the knee joint of an osteoarthritis model at week 8. Figure 22 shows the toxicity results of Fe-ATP self-assemblies confirmed in an osteoarthritis rat model. Figure 23 is a schematic diagram showing the bone regeneration effect of a 3D Fe-AMP self-assembly. Figure 24 is an SEM image showing a micropore structure in which 3D Fe-AMP self-assemblies are aggregated. Figure 25 is a diagram showing the magnetic targeting of a 3D Fe-AMP self-assembly. Figure 26 is a graph showing the AMP released over time from the Fe-AMP self-assembly. Figure 27 is the result of confirming via immunofluorescence imaging that osteogenic differentiation is promoted by AMP-degraded adenosine released from 3D Fe-AMP self-assemblies. Figure 28 is a Western blotting result confirming osteogenic differentiation by CD73-mediated AMP-degrading adenosine released from 3D Fe-AMP self-assemblies. Figure 29 is a C-Arm image of the femur to confirm fractures at 0, 2, 4, 6, and 8 weeks. Figure 30 is a micro CT image of the femur to identify a fracture at 8 weeks. Figure 31 shows H&E, osteocalcin, and TRAP staining images of a fracture. Figure 32 shows the results of confirming the toxicity of the in vivo 3D Fe-AMP self-assembly. Specific details for implementing the invention

[0048] Specific details of other embodiments are included in the detailed description and drawings.

[0049] The advantages and features of the present invention and the methods for achieving them will become clear by referring to the embodiments described below in detail together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below but may be implemented in various different forms. Unless otherwise specified in the following description, all numbers, values, and / or expressions representing components, reaction conditions, and the content of components in the present invention should be understood as being modified by the term "approximately" in all cases, as these numbers are approximations reflecting the various uncertainties of measurement that occur in obtaining these values ​​among essentially different things. Furthermore, where a numerical range is disclosed in this description, such range is continuous and, unless otherwise indicated, includes all values ​​from the minimum value of such range to the maximum value including said maximum value. Furthermore, where such range refers to an integer, unless otherwise indicated, it includes all integers including from the minimum value to said maximum value including said maximum value.

[0050] Additionally, when a range is described for a variable in the present invention, it will be understood that the variable includes all values ​​within the described range, including the described endpoints of the range. For example, the range "5 to 10" will be understood to include not only the values ​​5, 6, 7, 8, 9, and 10, but also any sub-ranges such as 6 to 10, 7 to 10, 6 to 9, 7 to 9, etc., and any values ​​between integers valid for the category of 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%” will be understood to include all integers including values ​​such as 10%, 11%, 12%, 13%, etc. and up to 30%, as well as any sub-range such as 10% to 15%, 12% to 18%, 20% to 30%, etc., and any value between valid integers within the stated range category such as 10.5%, 15.5%, 25.5%, etc.

[0052] FIG. 1 is a schematic diagram showing the formation of a self-assembly by iron ions and ATP according to one embodiment of the present invention. FIG. 2 is a schematic diagram showing the formation of a self-assembly by iron ions and AMP according to one embodiment of the present invention.

[0053] Referring to FIGS. 1 and 2, a therapeutic magnetic biomolecular-metal ion self-assembly complex according to the present embodiment may comprise an iron ion; and one or more ligands; wherein the ligand and the iron ion reversibly self-assemble or self-decompose, and the ligand and the iron ion self-assemble by a first bond to form a self-assembled body. In addition, in the therapeutic magnetic biomolecular-metal ion self-assembly complex, the self-assembly is performed by one or more of the metal ion and the ligand, and the self-assembly may be provided in multiple numbers, wherein adjacent self-assemblies are self-bonded by a second bond.

[0054] In this embodiment, the therapeutic magnetic biomolecule-metal ion self-assembly complex (hereinafter, biomolecule-metal ion self-assembly complex) is effective in treating diseases, etc., possesses magnetism, and includes a self-assembly in which a biomolecule and a metal ion are self-assembled, and the self-assembly can be provided in one or more units to form a complex.

[0055] The above 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, AEP (2-aminoethylphosphonic acid), TNA (Threose nucleic acid), GNA (glycol nucleic acid), HNA (1,5-anhydrohexitol nucleic acid), ANA (1,5-anhydroatritol nucleic acid), FANA (2'-deoxy-2'-fluoroarabino nucleic acid), and CeNA (cyclohexenyl nucleic acid). More specifically, the ligand may include one or more of AMP (adenosine monophosphate) and ATP (adenosine triphosphate).

[0056] The above ligand and the iron ion may self-bond by a first bond to form a self-assembly, and adjacent self-assemblies may self-bond by a second bond to form a biomolecular-metal ion self-assembly complex. In the biomolecular-metal ion self-assembly complex, 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.

[0057] In Fig. 1, iron ions (Fe 2+) can self-assemble through coordination bonding with the ligand ATP to form an Fe-ATP self-assembly. The Fe-ATP self-assemblies can self-assemble through one or more of hydrogen bonding and π-π interactions between adjacent Fe-ATP self-assemblies to form a therapeutic magnetic biomolecular-metal ion self-assembly complex according to one embodiment of the present invention.

[0058] Also, in Fig. 2, iron ions (Fe 2+ ) can self-assemble through coordination bonding with the ligand AMP to form an Fe-AMP self-assembly. The Fe-AMP self-assemblies can self-assemble through one or more of hydrogen bonding and π-π interactions between adjacent Fe-AMP self-assemblies to form a biomolecular-metal ion self-assembly complex according to one embodiment of the present invention.

[0059] The iron ion can form a metal complex by coordinating with the phosphate group of ATP or AMP and water (H2O). Additionally, the aromatic ring of the adenine portion of ATP or AMP can form π-π interactions. Furthermore, the nitrogen of the adenine portion and the water molecule bound to the iron ion can form hydrogen bonds. The biomolecular-metal ion self-assembled complex can be formed in the form of a complex containing one or more self-assembled bodies by self-bonding using one or more of the coordinate bonds, π-π interactions, and hydrogen bonds.

[0060] The self-assemblies or adjacent self-assemblies may self-assemble for a first time period or self-decompose for a second time period under physiologically relevant conditions. The first time period may be from 1 minute to 24 hours, and the second time period may be from 1 day to 90 days.

[0061] The above physiological conditions refer to temperature, pH, osmotic pressure, ionic strength, viscosity, and pseudo-biochemical parameters that are compatible with and / or typically present within the cells of live cultured yeast cells or mammalian cells. For example, in the case of human cells, these include a temperature of approximately 37°C, e.g., in the range of 35-39°C, a range of 1 kPa to 200 kPa, a pH of about 7, etc. Specifically, they mean conditions that are not toxic to the body or animal, and conditions that the body or animal can maintain.

[0062] The above self-assembly can be self-assembled for a first time period of 1 minute to 24 hours. If the time is less than 1 minute, the self-assembly cannot stably form the first bond or the second bond, which is problematic, and if it is 24 hours, it is sufficiently self-assembled. Therefore, maintaining the time beyond this period reduces efficiency. Specifically, the above first time period may be about 1 minute to 20 hours, or about 1 minute to 15 hours, or about 1 minute to 10 hours, about 1 minute to 5 hours, or about 1 minute to 2 hours, or about 1 minute to 30 minutes.

[0063] The above self-assembly is for the second period, 1 to 90 days It can self-decompose during the aforementioned time, and while self-decomposing during the aforementioned time, it can produce a stable and therapeutic effect by releasing iron ions or ligands. Specifically, the second time may be about 1 to 45 days, or about 1 to 30 days, or about 1 to 15 days, or about 1 to 10 days.

[0064] When the ligand is ATP (adenosine triphosphate), the therapeutic magnetic biomolecular-metal ion self-assembly complex may be provided in individual spherical shapes. Additionally, when the ligand is AMP (adenosine monophosphate), the therapeutic magnetic biomolecular-metal ion self-assembly complex may be provided as a three-dimensional aggregate having micropores formed by the aggregation of multiple self-assemblies.

[0065] Specifically, when the ligand is ATP, the biomolecular-metal ion self-assembly complex may have a spherical shape and be formed with a uniform diameter. The diameter of the biomolecular-metal ion self-assembly complex may be formed differently depending on one or more of the ligand concentration and the iron ion concentration. Specifically, when the iron ion concentration is high, the diameter of the self-assembly may increase. Additionally, when the ligand concentration is high, the diameter of the self-assembly may increase. In the biomolecular-metal ion self-assembly complex according to the present embodiment, when the ligand is ATP, the diameter of the biomolecular-metal ion self-assembly complex can be controlled by controlling the iron ion concentration and / or the ATP concentration.

[0066] When the ligand is AMP, the biomolecular-metal ion self-assembly complex may be provided in the form of a three-dimensional aggregate. Specifically, it may be provided in a form having a volume with multiple micropores inside. For example, even when the biomolecular-metal ion self-assembly complex uses AMP as the ligand, the size of the three-dimensional aggregate can be controlled by controlling the concentration of 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 by increasing 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 value of the cross-section diameter and height in the case of a cylinder, and the average value of the side length and height in the case of a polygon. The size of the above 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 µm to 30 cm, or about 200 µm to 30 cm, about 300 µm to 30 cm, or about 400 µm to 30 cm, or 500 µm to 30 cm, or 500 µm to 20 cm, or 500 µm to 10 cm.

[0067] The self-assembled body has paramagnetism, and the movement of the self-assembled body can be controlled by the application of an external magnetic field. Specifically, the biomolecular-metal ion self-assembled complex is provided in or outside the body, and the movement of the self-assembled body constituting the biomolecular-metal ion self-assembled complex can be controlled by the application of an external magnetic field. Accordingly, the biomolecular-metal ion self-assembled complex according to the present embodiment can control movement to a desired location, and specifically, even after injection into the body, the biomolecular-metal ion self-assembled complex can be moved to target cells or tissues to directly exert an effect.

[0068] The above self-assembly may be promoted to self-decompose under at least one of conditions including a chelating agent, strong acid conditions, and strong base conditions.

[0069] Under conditions including the above chelating agent, the chelating agent may be one or more of EDTA (ethylenediaminetetraacetic acid), bipyridyl, and ferrozine.

[0070] The chelating agent may form a bond with the iron ion instead of the ligand to promote the self-decomposition of the self-assembly. The chelating agent may be a substance with a higher affinity for the iron ion than the ligand, for example, it may be EDTA.

[0071] 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. Specifically, the pH of the strong acid condition may be 3 to 4, and the pH of the strong base condition may be 10 to 11. For example, the strong acid may be, for example, hydrochloric acid (HCl), and the strong base may be, for example, NaOH.

[0072] The above 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-decomposition of the self-assembled complex.

[0073] The self-assembled body may have paramagnetism. As the volume of the self-assembled body or the biomolecular-metal ion self-assembled complex increases, the magnetic moment may increase. Therefore, the larger the self-assembled complex is formed, the greater the attractive (or repulsive) force exerted by the magnetic field when a magnetic field is applied. Specifically, when a magnetic field is applied toward the biomolecular-metal ion self-assembled complex in vivo or in vitro, the movement of the biomolecular-metal ion self-assembled complex can be controlled by the magnetic field, and the larger the size of the biomolecular-metal ion self-assembled complex, the greater the movement speed of the biomolecular-metal ion self-assembled complex can be increased. Furthermore, since the biomolecular-metal ion self-assembled complex according to the present embodiment exhibits magnetism only when a magnetic field is formed on the biomolecular-metal ion self-assembled complex, the biomolecular-metal ion self-assembled complex can be easily controlled in vivo or in vitro.

[0075] The biomolecular-metal ion self-assembly complex according to the present embodiment can induce ferroptosis and can kill specific cancer cells.

[0076] Typically, cancer treatment relies on chemically synthesized drugs, and representative drugs such as doxorubicin, cisplatin, and methotrexate cause problems due to side effects affecting the heart, kidneys, and nerves. Additionally, while targeted therapies and immunotherapies are being developed and used to minimize damage to normal tissues during cancer treatment, it is difficult to utilize targeted therapies in cases like sarcoma because the target substances differ from patient to patient. Furthermore, immunotherapies are very expensive, making it very difficult to use them in actual treatment.

[0077] On the other hand, the biomolecular-metal ion self-assembly complex according to the present embodiment is non-toxic to living organisms and can target and move cancer cells by means of externally applied magnetism. In addition, the biomolecular-metal ion self-assembly complex can effectively kill cancer cells by inducing ferrotosis in them, and can be used stably without affecting surrounding normal cells or causing toxicity in the body. The ferrotosis can induce apoptosis in a manner different from apoptosis induced by oxidative damage to cellular phospholipids.

[0078] In the biomolecular-metal ion self-assembly complex according to the present embodiment, the self-assembly generates an excess amount of reactive oxygen species (ROS) under conditions containing hydrogen peroxide at a concentration of 10 μM or more, and the amount of reactive oxygen species generated may be proportional to the concentration of the self-assembly and the concentration of hydrogen peroxide added. Compared to cancer cells before the addition of the self-assembly and hydrogen peroxide, the amount of reactive oxygen species generated may be four times or more, and the death of cancer cells can be effectively induced through ferrotosis by the reactive oxygen species. Specifically, the concentration of hydrogen peroxide may be approximately 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.

[0079] Typically, normal cells have a hydrogen peroxide concentration of approximately 20 nM or less, while cancer cells exhibit a hydrogen peroxide concentration of approximately 10 μM or more, for example, 10 μM to 100 μM, which is higher than that of normal cells. Therefore, the biomolecular-metal ion self-assembly complex according to the present example self-decomposes only in cancer cells exhibiting high hydrogen peroxide concentrations, thereby Fe 2+ It emits, and the emitted Fe 2+ This can generate an excess amount of ROS through the Fenton reaction, which may lead to cancer cell death through ferroptosis.

[0080] The above reactive oxygen species oxidize cellular phospholipids and can induce apoptosis in cancer cells through ferrotosis by inhibiting GPX4 (glutathione peroxidase 4) or System xc-cystine / glutamate antiporter (Xc). GPX4 is an enzyme present in cells that repairs oxidized phospholipids, and GPX4 is regulated by System xc-cystine / glutamate antiporter (Xc).

[0081] The biomolecular-metal ion self-assembly complex according to the present embodiment contains iron ions, and the iron ions may be released by the self-decomposition of the self-assembly body constituting the biomolecular-metal ion self-assembly complex. The iron ions are biomaterials that react with hydrogen peroxide present in high concentrations within cancer cells to generate ROS through the Fenton reaction, and the ROS can damage cellular phospholipids through an oxidation reaction, thereby inducing ferrotosis in the cancer cells. Specifically, the ROS oxidizes the cellular phospholipids, and an excess of oxidized phospholipids is generated, leading to a decrease in GPX4 and Xc. When the oxidized phospholipids are generated in excess, and the condition becomes difficult to repair by GPX4, ferrotosis is induced in the cells.

[0082] If the ferrotosis of the aforementioned cells acts on organs other than cancer cells, cardiac side effects (such as cardiomyopathy) may occur; therefore, during the process of treating cancer by inducing ferrotosis using drugs such as artificially synthesized doxorubicin, it becomes problematic as it causes side effects even to normal cells surrounding the cancer cells.

[0083] On the other hand, the biomolecular-metal ion self-assembly complex according to the present embodiment can target local areas by an externally applied magnetic field, thereby selectively eliminating only cancer cells. The biomolecular-metal ion self-assembly complex is composed of a biomolecule and iron ions that are non-toxic to the body, so it is non-toxic to the body, has no side effects, and can be used continuously for a long period. In addition, the biomolecular-metal ion self-assembly complex can effectively act in cancer treatment by inducing ferrotosis specifically in cancer cells and killing cancer cells without affecting normal cells.

[0084] In the above biomolecular-metal ion self-assembly complex, the rate of self-decomposition of the self-assembly can increase under acidic conditions. Therefore, while the rate of decomposition is slow in normal cells under neutral conditions, the rate of decomposition can be rapid in cancer cells under acidic conditions. Furthermore, in cancer cells, ROS can be specifically generated by the above biomolecular-metal ion self-assembly complex due to relatively higher hydrogen peroxide levels and acidic conditions compared to normal cells. That is, in the biomolecular-metal ion self-assembly complex according to the present embodiment, since the rate of decomposition of the self-assembly occurs much faster in cancer cells than in normal cells, most of the Fenton reaction involving the decomposition of iron ions and ROS generation caused by the self-decomposition of the self-assembly acts on the cancer cells, so there is almost no effect on the normal cells.

[0085] In addition, compared to doxorubicin, a representative drug for conventional cancer treatment, the biomolecular-metal ion self-assembly complex according to the present embodiment enables localized targeting, has fewer side effects, exhibits similar efficacy in cancer cell death, and does not cause cardiac side effects.

[0086] In the biomolecular-metal ion self-assembly complex according to the present embodiment, the self-assembly has paramagnetism and its movement is controlled by the application of an external magnetic field, and by the application of the external magnetic field, the self-assembly moves to target cancer cells and can induce the death of cancer cells through ferrotosis.

[0087] When the above-mentioned self-assembly induces apoptosis of cancer cells through the above-mentioned ferrotosis, it can kill the cancer cells at a level similar to that of doxorubicin. Furthermore, while the above-mentioned self-assembly induces apoptosis of cancer cells through the above-mentioned ferrotosis, it may not exhibit toxicity to normal cells.

[0088] In the biomolecular-metal ion self-assembly complex according to the present embodiment, the self-assembly is used for cancer treatment, and the cancer may be 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 cancer, prostate cancer, skin cancer, tongue cancer, uterine cancer, stomach cancer, bone cancer, and blood cancer.

[0090] In a biomolecular-metal ion self-assembly complex according to another embodiment of the present invention, the self-assembly may self-assemble with an iron ion and a ligand through a first bond, and the self-assembly may self-assemble with neighboring self-assemblies through a second bond. At this time, the ligand includes ATP (adenosine triphosphate), and the self-assembly may release the ATP during self-decomposition to promote M2 polarization of macrophages through a P2Y1 receptor. Furthermore, the self-assembly promotes M2 polarization of macrophages distributed in the synovial membrane of a bone joint, and the M2 polarization of the macrophages may have anti-inflammatory activity of the synovial fluid of the bone joint.

[0091] Typically, arthritis treatment focuses on preventing inflammatory responses, and drugs that regenerate cartilage are virtually non-existent. Bone joints contain various types of cells, including MSCs, chondrocytes, macrophages, and fibroblasts; osteoarthritis worsens when the cytokine balance in the synovial fluid within the joint cavity is disrupted, or when pro-inflammatory cytokines that promote inflammation increase excessively compared to anti-inflammatory cytokines.

[0092] The above self-assembly contains ATP, a biomaterial, as a ligand, and the ATP can bind to various cell membrane receptors to control cell function. The types of cell membrane receptors that bind to the ATP can be controlled according to the concentration of ATP. The above macrophages can significantly affect the balance of cytokines in the synovial fluid within the bone joint. The synovial fluid within a bone joint where osteoarthritis has occurred contains about 0.5 nM of ATP, and when the ATP concentration is slightly increased through the above Fe-ATP self-assembly (e.g., several μM), it promotes M2 polarization through the P2Y receptor series to promote the secretion of anti-inflammatory cytokines, whereas when the above ATP concentration is very high (e.g., several mM), it promotes M1 polarization through the P2X receptor series to promote the secretion of pro-inflammatory cytokines.

[0093] Specifically, when the biomolecular-metal ion self-assembly complex according to the present embodiment is injected into a site where osteoarthritis has occurred, the self-assembly decomposes to release ATP, and the ATP is controlled at a concentration of 5-10 μM to promote M2 polarization of macrophages distributed in the synovial membrane of the bone joint, and the M2 polarization of the macrophages can have anti-inflammatory activity of the synovial fluid of the bone joint. Furthermore, when the ATP generated by the self-decomposition of the self-assembly controls the M2 polarization of the macrophages, it may not exhibit toxicity to normal cells while having anti-inflammatory activity of the synovial fluid of the bone joint. In addition, the self-assembly can prevent, improve, or treat osteoarthritis by maintaining an anti-inflammatory environment of the synovial fluid of the bone joint and protecting the osteochondral.

[0094] The self-assembled body has paramagnetism and its movement can be controlled by the application of an external magnetic field. Therefore, after injecting the biomolecular-metal ion self-assembled complex into the body, the self-assembled body moves to a target bone joint site by the application of an external magnetic field to protect the bone cartilage, thereby preventing, improving, or treating osteoarthritis.

[0096] A biomolecular-metal ion self-assembly complex according to another embodiment of the present invention comprises one or more self-assemblies, wherein the self-assemblies may be self-assembled by a first bond between an iron ion and a ligand. In this case, the ligand comprises AMP (adenosine monophosphate), and the biomolecular-metal ion self-assembly complex may be provided as a three-dimensional aggregate having micropores by aggregating a plurality of self-assemblies. The three-dimensional aggregate may have an average diameter of 500 μm to 10 cm. The self-assemblies may be used as a bone graft material to restore bone tissue by being implanted into a bone defect site in the form of the three-dimensional aggregates. For example, in a biomolecular-metal ion self-assembly complex formed by the three-dimensional aggregates, if the average diameter is less than 500 μm, it is too small to be used as a bone graft material and poses a problem, and if it exceeds 10 cm, the strength of the three-dimensional aggregates decreases and poses a problem.

[0097] The shape of the above three-dimensional aggregate can be formed into various three-dimensional structures by the mold used and may include a plurality of micropores inside. For example, the above three-dimensional aggregate may be provided with a network structure in which the micropores are interconnected three-dimensionally.

[0098] Clinically, treatment is very difficult when extensive bone defects occur in fractures of long bones, including the femur. Currently, various bone substitutes such as allogeneic, xenogeneic, and synthetic bone are used in conjunction with autogenous bone grafts; however, as the proportion of bone substitutes increases, the rate of bone resorption also increases, leading to lower therapeutic efficacy. Furthermore, autogenous bone grafts have the problem of causing many complications, such as delayed rehabilitation due to pain at the harvest site, bleeding, infection, and iatrogenic fractures.

[0099] In a biomolecular-metal ion self-assembly complex according to an embodiment of the present invention, the self-assembly can self-decompose to release AMP, and the released AMP promotes osteogenic differentiation of mesenchymal stem cells (MSCs) through signal transduction of adenosine receptors of mesenchymal stem cells (MSCs), and the osteogenic differentiation of mesenchymal stem cells (MSCs) can promote bone formation.

[0100] When the above biomolecular-metal ion self-assembly complex is implanted into a bone defect site, it acts as an implant material for the bone defect site and can simultaneously release AMP through the self-decomposition of the self-assembly. The AMP released from the self-assembly can fill the bone defect site and promote MSC attachment and osteogenic differentiation of MSCs.

[0101] In addition, the self-assembly according to the present embodiment has paramagnetism and its movement can be controlled by the application of an external magnetic field. When the self-assembly is implanted into a bone defect site in the form of the three-dimensional aggregate, the self-assembly can move in one or more of the same direction and the other direction by the application of the external magnetic field.

[0102] The above-described self-assembly is implanted into a bone defect site, and host cells can attach to the surface of the self-assembly to form a skeleton. Additionally, the self-assembly has paramagnetism and its movement can be controlled by the application of an external magnetic field. For example, when the self-assembly is implanted into a bone defect site, the self-assembly can move by the application of the external magnetic field, and at this time, the host cells attached to the surface of the self-assembly can also move together.

[0103] The above self-assembly is implanted into the bone defect site, and the self-assembly self-decomposes for 1 to 70 days The above AMP can be released during this period. The AMP released from the self-assembly can be broken down into adenosine on the surface of the host cell and can promote signaling of adenosine receptors on the surface of the host cell. For example, the adenosine receptor may include an adenosine A2B receptor.

[0104] When the above-mentioned self-assembly is implanted into a bone defect site to regenerate the defective bone tissue, it can promote bone regeneration to a level similar to that of calcium-phosphate cement, a bone defect graft material. Furthermore, while the above-mentioned self-assembly is implanted into the bone defect site to regenerate the defective bone tissue, it may not exhibit toxicity to normal cells.

[0106] The therapeutic magnetic biomolecular-metal ion self-assembly complex according to the present embodiment may be a self-assembly complex formed by the bonding of monomeric self-assemblies, and said self-assemblies may include an iron ion and a ligand that self-assembles with said iron ion through a first bond. Additionally, said self-assemblies may form the therapeutic magnetic biomolecular-metal ion self-assembly complex by having neighboring self-assemblies self-assemble through a second bond.

[0107] The above self-assembly can self-decompose due to the surrounding environment of the self-assembly, and the speed of self-decomposition can also be controlled.

[0108] The above ligand may include one or more of AMP (adenosine monophosphate) and ATP (adenosine triphosphate). For example, when the self-assembly undergoes self-decomposition, the iron ion or the ligand may be released. Additionally, by controlling the rate of self-decomposition of the self-assembly, the rate at which the iron ion or the ligand is released can be controlled.

[0109] The size of the above-mentioned therapeutic magnetic biomolecular-metal ion self-assembly complex can be controlled by controlling the manufacturing method, for example, the concentration of the material containing iron ions and the concentration of the material containing the ligand.

[0110] The self-assembled body has paramagnetism and can be controlled in movement by an externally applied magnetic field, so the movement of the biomolecular-metal ion self-assembled complex can be controlled outside the body, and even when the biomolecular-metal ion self-assembled complex is implanted inside the body, the position and movement speed of the biomolecular-metal ion self-assembled complex can be controlled by an externally applied magnetic field.

[0111] If the ligand is one or more of AMP and ATP, the self-assembly induces the death of cancer cells through ferrotosis, and if the ligand is ATP, the self-assembly can prevent, improve, or treat osteoarthritis. In addition, if the ligand is AMP, the self-assembly can be implanted into a bone defect site to regenerate the defective bone tissue.

[0113] Examples and comparative examples of the present invention are described below. However, the following examples are merely preferred embodiments of the present invention, and the scope of the present invention is not limited by the following examples.

[0115] Manufacture of self-assemblies

[0117] 1. Ingredients

[0118] Adenosine-5'-monophosphate disodium salt (CAS: 4578-31-8, Alfa Aesar)

[0119] Adenosine 5'-triphosphate disodium salt (CAS: 51963-61-2, Daejeong reagent)

[0120] Ferric chloride (Iron(II) chloride, 97%, Sigma-Aldrich, 25 g, cat. No. 372870)

[0121] PMMA (poly(methyl methacrylate), diameter 125 to 150 μm, Bangs Laboratories, BB05N)

[0123] 2. Preparation of Fe-ATP Self-Assemblies

[0125] Preparation Example 1

[0126] To synthesize Fe-ATP self-assemblies, FeCl2 solution and ATP (adenosine triphosphate) solution were prepared using deionized water (DI water). The FeCl2 solution was prepared by adding 25.35 mg of ferric chloride (Iron(II) chloride, 97%, Sigma-Aldrich, 25 g, cat. No. 372870) to 10 mL of deionized water, and the ATP solution was prepared by adding 110.23 mg of adenosine-5'-triphosphate disodium salt (CAS: 51963-61-2, Daejeong Reagent) to 10 mL of deionized water.

[0127] A mixed solution was prepared by adding 1 mL of 20 mM FeCl2 solution to a reactor containing 1 mL of 20 mM ATP solution and mixing it using a vortex mixer (Vortex Genie 2, Scientific Industries). The prepared mixed solution was maintained at room temperature for 12 hours, and Fe 2+Fe-ATP self-assemblies were prepared by inducing spontaneous assembly between Fe and ATP. A solution containing Fe-ATP self-assemblies was centrifuged at 10,000 rpm for 5 minutes. To remove unreacted reagents, the supernatant was discarded, and the solution was centrifuged twice after adding deionized water. After washing, 112.6 mg of particulate Fe-ATP self-assemblies were dispersed in 2 mL of deionized water. The prepared Fe-ATP self-assemblies are shown in Table 1.

[0129] Preparation Example 2

[0130] Fe-ATP self-assemblies were prepared in the same manner as in Preparation Example 1, except that when preparing the mixed solution, 1 mL of 1 mM FeCl2 solution was used in a reactor containing 1 mL of 1 mM ATP solution and the prepared mixed solution was reacted at room temperature for 30 minutes, and the results are shown in Table 1.

[0132] Preparation Example 3

[0133] Fe-ATP self-assemblies were prepared in the same manner as in Preparation Example 1, except that when preparing the mixed solution, 1 mL of 0.1 mM FeCl2 solution was used in a reactor containing 1 mL of 0.1 mM ATP solution and the prepared mixed solution was reacted at room temperature for 30 minutes, and the results are shown in Table 1.

[0135] Category 1 ATP solution concentration FeCl2 solution concentration average particle size Preparation Example 1 20mM 20mM 600nm Preparation Example 2 1mM 1mM 150nm Preparation Example 3 0.1mM 0.1mM 70nm

[0137] 3. Preparation of Fe-AMP Self-Assembly

[0139] Preparation Example 4

[0140] To synthesize the Fe-AMP self-assembly, 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 ferric chloride (Iron(II) chloride, 97%, Sigma-Aldrich, 25 g, cat. No. 372870) to 10 mL of deionized water, and the AMP solution was prepared by adding 78.24 mg of AMP (adenosine monophosphate) to 10 mL of deionized water.

[0141] A mixed solution was prepared by adding 1 mL of 20 mM FeCl2 solution to a reactor containing 1 mL of 20 mM AMP solution and mixing it using a vortex mixer (Vortex Genie 2, Scientific Industries). The prepared mixed solution was maintained at room temperature for 12 hours, and Fe 2+ Fe-AMP self-assemblies were prepared by inducing spontaneous assembly between and AMP. A solution containing the Fe-AMP self-assemblies was centrifuged at 10,000 rpm for 5 minutes. To remove unreacted reagents, the supernatant was discarded, and the solution was centrifuged after adding deionized water, repeating this process twice. After washing, 89.24 g of particulate Fe-AMP self-assemblies were dispersed in 2 mL of deionized water.

[0143] 4. Fe3O4 Synthesis

[0145] Preparation Example 5

[0146] 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 mixed solvent 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 iron-oleate complex contained in the upper hexane layer was washed with deionized water and the hexane was evaporated to obtain a dried iron-oleate complex. Subsequently, the iron-oleate complex was mixed with 0.14 g of oleic acid and 5 g of 1-octadecene. This mixed solution was vigorously stirred at 320°C for 30 minutes and then cooled to 25°C. The synthesized Fe3O4 nanoparticles from the cooled mixed solution were repeatedly washed with ethanol and centrifuged to produce Fe3O4 nanoparticles with an average diameter of 10 nm.

[0148] Basic performance evaluation method for self-assemblies

[0150] 1. Confirmation of Fe or ATP release characteristics from Fe-ATP self-assemblies

[0152] Fe and ATP release characteristics were verified using Fe-ATP self-assemblies.

[0153] The FeCl2 solution was prepared by adding 25.35 mg of ferric chloride (Iron(II) chloride, 97%, Sigma-Aldrich, 25 g, cat. No. 372870) to 10 mL of deionized water, and the ATP solution was prepared by adding 110.23 mg of adenosine-5'-triphosphate disodium salt (CAS: 51963-61-2, Daejeong Reagent) to 10 mL of deionized water.

[0154] A mixed solution was prepared by adding 5 mL of 20 mM ATP solution and 5 mL of 20 mM FeCl2 solution to Falcon tubes and mixing for 1 minute using a Vortex Genie 2 (Scientific Industries). The prepared mixed solution was maintained at room temperature for 12 hours. Subsequently, it was 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 the deionized water, the solution was centrifuged once again at 10,000 rpm for 5 minutes, the supernatant was removed, and the solid Fe-ATP self-assembled material was recovered. The removed supernatant was used to measure ATP loading efficiency by subtracting the amount of unloaded ATP from the amount of initially added ATP via HPLC. Add 1 mL of PBS solution (pH 7.4 or pH 5.5) to the recovered Fe-ATP self-assemblies, and place them in a dialysis bag (SnakeSkin). TM It was transferred to Dialysis Tubing, 7kDa MWCO.

[0155] A dialysis bag containing Fe-ATP self-assemblies was placed in a vial containing 4 mL of PBS solution (pH 7.4 or pH 5.5) and stored at room temperature. From the vial containing the dialysis bag with Fe-ATP self-assemblies, 40 μL of the supernatant was collected on days 1, 3, 5, and 7, respectively, and the concentration of released Fe or ATP was measured and calculated. Here, PBS solution was added to the vial to maintain a consistent total volume.

[0156] Cumulative ATP release profiles were obtained using an HPLC (Arc HPLC Core System, Waters) equipped with an analytical column (XBridge BEH C18, 130 Å, 4.6 mm x 250 mm, particle size 5 μm). 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 is a PBS solution.

[0157] 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 which the mobile phase A / B = 100 / 0 (v / v) was maintained constant for another 1 min. The flow rate was 1.2 mL / min with isosolvent, the injected sample volume was 20 μL, and UV-vis absorbance was monitored at 260 nm. Additionally, to obtain cumulative Fe emission profiles at various pH values, Fe analysis (QuantiChrom™ Iron Assay Kit, Bioassay systems) was used (Figs. 7 and 14 below).

[0159] 2.1. Verification of AMP Emission Characteristics from Fe-AMP Self-Assembly

[0161] The AMP emission characteristics were verified using an Fe-AMP self-assembly.

[0162] A cumulative AMP release profile was obtained to confirm continuous AMP release for AMP-induced fracture healing. To prepare Fe-AMP self-assemblies, adenosine-5'-monophosphate disodium salt (CAS: 4578-31-8, Alfa Aesar) was used instead of adenosine-5'-triphosphate disodium salt (CAS: 51963-61-2, Daejeong reagent), and Fe-ATP self-assemblies were recovered using the same method as the prepared samples to confirm Fe or ATP release characteristics.

[0163] From vials containing dialysis bags equipped with Fe-AMP self-assemblies, 400 μL of the supernatant was collected on days 1, 3, 7, 14, and 21, respectively, 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 Fig. 26 below).

[0165] 3. IVIS L-012 ROS Signal Analysis

[0167] The luminescent probe L-012 (Wako Chemical, Korea) was dissolved in ultra-pure H2O and prepared in a fresh state immediately before the experiment. 25 mg / kg was administered subcutaneously to the backs of mice at a 50 µl injection dose.

[0168] Chemiluminescence (CL) emission evaluation was initiated immediately after L-012 injection using indiGO™ software and IVIS (In vivo Imaging System, NightOWL II LB 983, BERTHOLD Technologies GmbH, Germany). Mice were exposed for 20 seconds each time to collect sufficient CL emission data. CL emitted from the mouse skin was recorded and visualized in real time, and the CL emitted from the region of interest was quantified as total flux (photons / second).

[0170] Evaluation method for cancer cell death, osteoarthritis, and bone defect treatment using self-assemblies

[0172] Experimental Example 1 (Cancer cell death, osteosarcoma model)

[0174] To synthesize Fe-ATP self-assemblies, FeCl2 and ATP (adenosine triphosphate) solutions 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 solutions of various concentrations. 5 mL of 20 mM FeCl2 solution and 5 mL of 20 mM ATP (adenosine triphosphate) solution were added at equal concentrations and mixed for 1 minute using a vortex mixer (Vortex Genie 2, Scientific Industries) to prepare mixed solutions. The mixed solutions were maintained at room temperature for 12 hours to induce self-assembly and produce Fe-ATP self-assemblies. The solutions containing the Fe-ATP self-assemblies were 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 solution was injected into mice.

[0175] 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-assemblies in osteosarcoma, they were compared with PBS (Phosphate-buffered saline), ATP (Daejeong reagent, CAS: 51963-61-2), DOX (Doxorubicin), Fe-ATP, Fe-ATP (mag), Fe3O4, and Fe3O4 (mag) as shown in Table 2 below. At this time, 5 mg / kg of Fe-ATP and 7 mg / kg of Fe3O4 were injected, respectively.

[0177] division particular particle size Magnetic field application PBS (No treatment) Welgene, LB 204-02 - x DOX AD mycin, Boryung Pharmaceutical, CAS #23214-92-8 - x Fe-ATP Synthesized Fe-ATP self-assembly 600nm x Fe-ATP (mag) Synthesized Fe-ATP self-assembly 600nm o Fe3O4 Preparation Example 5 10nm x Fe3O4(mag) Preparation Example 5 10nm o

[0179] Before performing the surgery, the mice were anesthetized by injecting a mixture of 50-70% medical oxygen and 2% isoflurane (CAS # 26675-46-7) at a rate of 2 L / min. After making a skin incision on the knee area of ​​the mice, the knee joint was identified, and the patellar tendon was exposed and then incised to expose the upper part of the tibia. Subsequently, a hole was made in the exposed area with a needle, and pre-prepared KHOS osteosarcoma cells (1 x 10⁷ / mL, 100 µL) were injected into the bone marrow, after which the skin was sutured. After injecting osteosarcoma cells into mice, to prevent infection, the antibiotic enrofloxacin (Baytril, Bayer, CAS # 93106-60-6) was diluted fivefold in physiological saline and injected once a day for 5 days after surgery, and the analgesic ketoprofen (Ketopro inj, Unibio Co. Ltd, CAS # 22071-15-4) was administered for 1 day after surgery to relieve pain.

[0180] Before injecting the Fe-ATP self-assembly, mice were anesthetized by inhalation by injecting a gas mixture of 2% isoflurane with oxygen at a rate of 2 L / min. Four weeks after surgery, the drug was administered six times over a total of six weeks at seven-day intervals according to the groups listed in Table 2. 20 μL of Fe-ATP self-assembly was injected into each tumor site, and 2 mg / kg of doxorubicin (Doxorubicin hydrochloride, AD mycin, Boryung Pharmaceutical, CAS # 23214-92-8) was injected to compare the effect with that of a conventional chemoagent. The Fe3O4 group was engineered to inject 5 mg / kg of Fe ions into the mouse, and the same volume of 20 μL was injected. Here, the injection amount of Fe-ATP autoassembly was set to 5 mg / kg (autoassembly weight / mouse weight), and the injection amount of Fe3O4 was set to 7 mg / kg (autoassembly weight / mouse weight). As a comparative example, the PBS groups were each injected with an equal volume of 20 μL of PBS (pH 7.4). To confirm magnetic targeting, a 270 mT circular neodymium magnet (diameter 5 mm, height 2 mm) was fixed at the injection site using tegadam and surgical tape in the groups labeled (mag) in each category.

[0181] After 6 weeks, the mice were euthanized using CO2, and tumor tissue samples were collected from the leg area and histological examination was performed (histology, Department of Pathology, Korea University, Seoul, Korea).

[0183] Experimental Example 2 (Osteoarthritis Model)

[0184] 1 mg of Fe-ATP self-assembled particles prepared in the same manner as in Experimental Example 1 was dispersed in 10 mL of deionized water, and 20 μL was injected into rats.

[0185] The mice used in this experiment were 8-week-old Sprague-Dawley rats, and to confirm the efficacy of Fe-ATP self-assembly in osteoarthritis, it was compared with PBS (Phosphate-buffered saline) (No treatment), Fe-ATP, and Fe-ATP (mag) as shown in Table 3 below. Here, the injection dose of Fe-ATP self-assembly was 8 μg / kg (weight of self-assembly / weight of rat). Since the synovial fluid of one rat is approximately 100 μL, 2 μg of Fe-ATP self-assembly was dissolved in 20 μL of PBS and injected. Referring to the ATP release profile using Fe-ATP self-assembly (see Fig. 14), at room temperature, approximately 7% of ATP is released from the Fe-ATP self-assembly over 1 day, and approximately 28% of ATP is released over 7 days. Therefore, when 8 μg / kg of Fe-ATP self-assembly (weight of self-assembly / weight of rat) is injected into a rat, the ATP concentration in the rat's synovial fluid becomes 5–8 μM, which stimulates M2 macrophages.

[0187] division particular particle size Magnetic field application PBS (No treatment) Welgene, LB 204-02 - x Fe-ATP Synthesized Fe-ATP self-assembly 600nm x Fe-ATP (mag) Synthesized Fe-ATP self-assembly 600nm o

[0189] Experiments were performed on rats in the following order, and the animals were subjected to a 7-day quarantine / acclimation process prior to the experiment.

[0190] First, 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) were intramuscularly injected into rats, and mice were anesthetized by inhalation by injecting a gas mixed with 2% isoflurane in oxygen at a rate of 2 L / min. To prevent infection, the antibiotic enrofloxacin (Baytril, Bayer, CAS # 93106-60-6) was diluted fivefold in physiological saline and administered at a dose of 5 mg / kg once a day for 5 days after surgery, and to relieve pain, the analgesic ketoprofen (Ketopro inj, Unibio Co. Ltd, CAS # 22071-15-4) was administered intradermally at a dose of 5 mg / kg for 1 day after surgery.

[0191] To perform anterior cruciate ligament transection (ACLT), the skin was incised at the knee, the knee joint was visualized, and the patellar tendon was set aside to expose the anterior cruciate ligament. The exposed anterior cruciate ligament was cut with ophthalmic scissors from the center to one-third of the way down, and a Lachman test was performed to confirm the transection. After rinsing the knee with normal saline, treatment according to Table 3 was administered. For the groups corresponding to Table 3, treatment was performed by injecting into the synovial fluid within the joint cavity. For each group, the drug was injected eight times over a total of eight weeks at seven-day intervals. 20 μL of Fe-ATP self-assembly and 20 μL of PBS were injected in equal volumes. For the Fe-ATP (mag) group, 20 μL of Fe-ATP autoassembly was injected in the same manner, and a 270 mT circular neodymium magnet (diameter 8 mm, height 2 mm) was fixed to the joint area with surgical tape to confirm the magnetic targeting of the Fe-ATP autoassembly. After performing each treatment, the skin of the knee area was sutured again, and after 8 weeks, the following plain radiography, micro-computed tomography (Micro-CT), and histological analysis were performed.

[0192] To perform plain radiography (at 0, 2, 4, 6, and 8 weeks), general anesthesia was administered by intramuscular injection of 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). Immediately after surgery, the ACLT and the presence of surrounding fractures were confirmed using plain radiography equipment (Cios Alpha, Siemens). Subsequently, plain radiography was performed at 2-week intervals for 8 weeks to monitor the radiological course, and the results were scored using the Kellgren-Lawrence score.

[0193] To confirm Micro-CT (8 weeks) and histology (8 weeks), general anesthesia was administered by intramuscular injection of 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), followed by euthanasia using CO2 gas. Knee cartilage was harvested and fixed in a 4% paraformaldehyde solution. A micro CT scan (Genoss, Suwon, Korea) was performed to evaluate the regenerative status of the cartilage.

[0195] Experimental Example 3 (Bone defect model)

[0197] A 3D Fe-AMP self-assembly was fabricated in the form of a scaffold having micropores. For macroporous bone scaffolds containing 3D Fe-AMP self-assembled micropores, a leaching method using PMMA (poly(methyl methacrylate)) was employed. First, 300 mg of PMMA (diameter 125–150 μm, Bangs Laboratories, BB05N) was filled into a cylindrical polyethylene mold (8 mm diameter). 200 μL of a 2 M FeCl2 solution dissolved in deionized water was added to the mold and gently mixed using a vortex mixer for 5 minutes. Subsequently, 200 μL of a 2 M AMP solution dissolved 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 maintained at 25°C for 1 day. Afterward, the 3D Fe-AMP aggregates were separated from the mold, and the mixture was subjected to vibration conditions (100 rpm) for 3 days to leach the PMMA. It was impregnated in 50 mL of dichloromethane (DCM). The DCM was replaced every 24 hours. After 3 days, the 3D Fe-AMP aggregates, in which 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 pieces 1 cm or 0.8 cm high and sterilized using UV irradiation for 30 minutes before use for bone treatment.

[0198] The rabbits used in this experiment were 16 to 20-week-old New Zealand White males. To confirm the efficacy of 3D Fe-AMP aggregates in treating bone defects, the rabbits were treated by classifying them into a control group (No treatment, control), Short Fe-AMP, Short Fe-AMP(mag), Long Fe-AMP, and Ca-phosphate, respectively, as shown in Table 4 below. Here, Short Fe-AMP is a 0.8 cm Fe-AMP aggregate, Long Fe-AMP is a 1 cm Fe-AMP aggregate, and (mag) is a 270 mT circular neodymium magnet (diameter 8 mm, height 2 mm) fixed with surgical tape to confirm magnetic targeting.

[0200] division particular size Magnetic field application PBS (No treatment) Welgene, LB 204-02 - x Short Fe-AMP Synthesized Fe-AMP self-assembly 0.8cm x Short Fe-AMP(mag) Synthesized Fe-AMP self-assembly 0.8cm o Long Fe-AMP Synthesized Fe-AMP self-assembly 1cm x Ca-phosphate SN Biologics, NeoBone 1cm x

[0202] Experiments were performed on rabbits in the following order, and the animals were subjected to a 7-day quarantine / acclimation process prior to the experiment.

[0203] In the preparation of a wide bone defect model (0-8 weeks), prior to surgery, deep anesthesia (xylazine + alfalaxone + isoflurane) was administered, and antibiotics (enrofloxacin) and analgesics (ketoprofen) were given. The mice were anesthetized by intramuscular injection of xylazine (5 mg / kg IM) and alfaxalone (3 mg / kg IV), and after intradermal injection of the antibiotic enrofloxacin (5 mg / kg SC) and the analgesic ketoprofen (5 mg / kg IM), the mice were anesthetized by inhalation by injecting a gas mixed with 2% isoflurane in oxygen at a rate of 2 L / min.

[0204] After confirming that cardiac anesthesia was complete, an incision of approximately 7 cm was made in the rabbit's femur, and the femur was reached via an anterolateral approach. Subsequently, the location for creating a 1 cm bone defect on the femoral shaft was marked, and a hole was created at the resection site using a metal pin. Using a surgical electric saw (Colibri II, Depuy Synthes), partial resection was performed only on the portion of the bone resection site that would be covered by a metal plate. A 6-hole 2.7 locking compression plate (LCP 2.7 straight (6 holes, L 58 mm) - Depuy Synthes) was positioned to secure three metal screws proximal to two distal to the 1.5 cm bone defect, and the three proximal and two distal screws were carefully secured to prevent fracture of the rabbit's bone. Finally, the partially resected bone resection site was carefully completed using the surgical electric saw (Colibri II, Depuy Synthes). At this time, the rabbit's bone is highly brittle and breaks easily, so the process was carried out with caution.

[0205] After inserting 3-D Fe-AMP aggregates or bone graft material (main component calcium triphosphate, NeoBone, SN Biologics, Suwon, Republic of Korea) into the bone defect, fascial and skin suturing were performed. For the Short Fe-ATP (mag) group, to control magnetic movement for cell recruitment, 270 mT circular neodymium magnets (diameter 20 mm, height 2 mm) were positioned alternately on the upper and lower sides of the surgical site on a daily basis, changing positions three times, and secured with tegadam and surgical tape. Subsequently, membrane formation and maturation were waited for 8 weeks, and the antibiotic enrofloxacin (5 mg / kg SC) and the analgesic ketoprofen (5 mg / kg IM) were injected for 3 days post-surgery. To evaluate the experimental results, the following analyses of plain radiography, micro-computed tomography (Micro-CT), and histology (8 weeks) were performed.

[0206] Plain radiographs (at 0, 2, 4, 6, and 8 weeks) were performed using radiographic equipment (Cios Alpha, Siemens). Prior to the procedure, anesthesia was administered via intramuscular injection of Xylazine 5 mg / kg IM and Alfaxalone 3 mg / kg IV. Immediately after the first surgery, plate fixation and the occurrence of surrounding fractures were checked. The clinical course was then monitored for 4 weeks until guiding membrane maturation; if any abnormalities were observed, a plain radiograph was performed 2 weeks after the first surgery to monitor the radiological course. Subsequently, plain radiographs were performed at 2-week intervals for a total of 8 weeks to monitor the radiological course, which was scored using the RUST score. At 8 weeks post-surgery, bone union was evaluated based on the union of at least 3 out of 4 cortical bones in two plain radiographs: anterior (anteroposterior) and lateral (lateral).

[0207] Micro-CT scans (at 0 and 8 weeks) were performed at the institution (Genoss, Suwon, Korea). 8 weeks after surgery, rabbits were sacrificed (KCl) to obtain the femur, fixed in a 4% paraformaldehyde solution, and then micro-CT scans were performed to evaluate the volume of the reconstructed bone using 3D software (provided by Genoss).

[0208] Histological examinations (at 8 weeks) were performed after administering cardiac anesthesia prior to euthanasia. Euthanasia was induced using potassium chloride (dose: 2 mmol / kg, IV). After anesthetizing patients with Xylazine (5 mg / kg IM) and alfaxalone (3 mg / kg IV) at double the prescribed doses, potassium chloride (dose: 2 mmol / kg, IV) was injected to induce euthanasia. After inducing euthanasia, it was confirmed that cardiac arrest had definitely occurred. Organ toxicity and peribular muscle tissue biopsy were performed by H&E (Department of Pathology, Korea University, Seoul, Korea), and bone immunohistochemistry was performed by IHC (Osteocalcin, Genoss, Suwon, Korea).

[0210] Basic performance of the self-assembly and evaluation results of cancer cell death, osteoarthritis, and bone defect treatment using the self-assembly

[0212] In the following, using FIGS. 3 to 32, the basic performance of a self-assembly manufactured according to an embodiment of the present invention and the evaluation results of cancer cell death, osteoarthritis, and bone defect treatment using the self-assembly are presented.

[0214] Figure 3 shows an SEM image of the Fe-ATP self-assembly and the results of measuring paramagnetism. The paramagnetism of the above Fe-ATP self-assembly was measured using a vibrating sample magnetometry (VSM). Figure 3 is an SEM image of the Fe-ATP self-assembly prepared according to Preparation Example 1, and it was confirmed that it was prepared in the form of spherical particles of uniform size with an average diameter of 600 nm.

[0215] The Fe-ATP self-assembly is Fe 2+ It was confirmed that iron ions and ATP primarily form self-assemblies through coordination bonding, and that the prepared Fe-ATP self-assemblies aggregate due to hydrogen bonding or π-π interactions between neighboring Fe-ATP self-assemblies. Furthermore, it was confirmed that the Fe-ATP self-assemblies prepared in an aggregated form were spherical with a nearly uniform average diameter. Additionally, after forming a magnetic field using a circular neodymium magnet of 270 mT (diameter 8 mm, height 2 mm), the Fe-ATP self-assemblies moved. That is, it was confirmed that the Fe-ATP self-assemblies according to Preparation Example 1 possess paramagnetism, a reversible magnetic property, and thereby exhibit movement when attracted by an external magnet.

[0216] Figure 4 is an SEM image of Fe-ATP self-assemblies with average diameters of 150 nm and 70 nm, respectively, according to an embodiment of the present invention. Figure 4 shows Fe-ATP self-assemblies prepared according to Preparation Examples 2 and 3, confirming that the diameter of the self-assemblies is controlled by the concentration of iron ions and the concentration of ATP. When prepared in the same manner, but with the iron ion concentration set to 1 mM and the ATP concentration set to 1 mM as in Preparation Example 2, the average diameter of the Fe-ATP self-assemblies was 150 nm, and when the iron ion concentration set to 0.1 mM and the ATP concentration set to 0.1 mM as in Preparation Example 3, the average diameter of the Fe-ATP self-assemblies was 70 nm. That is, the average diameter of the Fe-ATP self-assemblies could be increased by increasing the iron ion concentration or by increasing the ATP concentration.

[0217] Figure 5 is a TEM image of an Fe-AMP self-assembly. Figure 5 is an Fe-AMP self-assembly prepared using iron ions and AMP according to Preparation Example 4. It was confirmed that the Fe-AMP self-assembly is provided in a 3D form having micropores, in a form where multiple units are aggregated. That is, it was confirmed that multiple Fe-AMP self-assemblies are aggregated and micropores are formed between the aggregated Fe-AMP self-assemblies.

[0219] Figures 6 to 12 below show the experimental results related to Experimental Example 1.

[0221] Figure 6 is a schematic diagram illustrating the induction of ferroptosis in cancer cells using Fe-ATP self-assemblies. Figure 6 relates to Experimental Example 1, in which Fe-ATP self-assemblies implanted in vivo could be moved by applying magnetic force from outside the body. Furthermore, when moving the Fe-ATP self-assemblies, they could be transported to targeted cancer cells, after which the Fe-ATP self-assemblies self-decomposed to produce iron ions (Fe 2+Ferrotosis of cancer cells was induced by continuously releasing ). That is, the Fe-ATP self-assembly according to the present embodiment can target specific cancer cells by self-decomposition and by shifting the position of the Fe-ATP self-assembly by a magnetic field. Therefore, the Fe-ATP self-assembly can enable an iron ion-mediated Fenton reaction for cancer treatment using ferotosis.

[0222] Figure 7 is a graph showing the amount of iron ions released from the Fe-ATP self-assembly at pH 5.5 or pH 7.4, measured using an iron assay kit. The Fe released from the Fe-ATP self-assembly in Experimental Example 1 2+ Before confirming the effect, Fe in Fe-ATP self-assemblies using PBS similar to biological fluids 2+ The release amount was confirmed. At both pH 5.5 and pH 7.4, the Fe-ATP self-assembly self-decomposed to produce iron ions, Fe 2+ It was confirmed that [it] was released. In addition, Fe [it] at pH 5.5 was higher than at pH 7.4. 2+ It was confirmed that it was released faster and in greater quantities. In other words, Fe 2+ The release of is accelerated under relatively acidic conditions, and by changing the surrounding conditions of the Fe-ATP self-assembly, Fe 2+ It was confirmed that the release rate and release amount of can be controlled. In the Fe-ATP self-assembly according to the present embodiment, Fe 2+ The amount of release can be varied by designing the Fe-ATP self-assembly in various ways.

[0223] Figure 8 shows the intracellular ROS concentrations measured by DCFDA staining according to each condition. Experiments under each condition were performed on cells prior to experiments in mice according to Experimental Example 1 below. As shown in Table 5 below, each condition was classified using KHOS cancer cells into a control group treated only with PBS, treated only with DOX, treated with Fe-ATP, treated only with ATP, and treated with FeCl2. Here, in the case of FeCl2, it dissolves and Fe 2+ and Cl - Since it is broken down into, the Fe released from Fe-ATP 2+ It was added for comparison. It was confirmed that when Fe-ATP self-assemblies were used, ROS production was promoted 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 production was promoted more than when ATP alone was used. In other words, this is considered to be an effect caused by iron ions generated by the autolysis of Fe-ATP self-assemblies.

[0224] It was confirmed that the Fe-ATP self-assembly of the present invention generates ROS more effectively than toxorubicin (DOX), which is commonly used in the treatment of cancer cells. Figure 8 shows cancer cells, in which the concentration of hydrogen peroxide inside the cancer cells is higher (10μM-100μM) than that of normal cells (20nM). Excess ROS is generated through the Fenton reaction with Fe2+ released by self-decomposition from the Fe-ATP self-assembly according to the present embodiment, resulting in cancer cell death through ferroptosis.

[0226] division particular PBS (No treatment) Welgene, LB 204-02 DOX AD mycin, Boryung Pharmaceutical, CAS #23214-92-8 Fe-ATP Synthesized Fe-ATP self-assembly (600 nm) ATP Daejeong Reagent, CAS: 51963-61-2 FeCl2 Iron(II) chloride, Sigma-Aldrich, cat. No. 372870

[0228] Figure 9 shows the results of analyzing the IVIS L-012 ROS signal 2 hours after substance injection. Figure 9 pertains to Experimental Example 1, confirming that the Fe-ATP self-assembly significantly stimulates ROS generation in vivo. The control group, PBS, produced almost no ROS, while Fe3O4 showed results similar to those obtained with PBS alone. This is because Fe3O4 is not degraded in vivo, and Fe 2+ It is believed that this is because it does not release ROS. In addition, the Fe-ATP self-assembly according to this embodiment showed a higher effect than doxorubicin (DOX). While doxorubicin (DOX), which is used in conventional cancer treatment, has almost no effect on ROS release in a short period of time, it was confirmed that the Fe-ATP self-assembly is more effective in killing cancer cells by generating an excessive amount of ROS even in a short period of time.

[0229] Fig. 10 shows the continuous Fe of a magnetically targeted Fe-ATP self-assembly. 2+ This is the result of confirming the ferrotosis of cancer cells through release. It was confirmed that the Fe-ATP self-assembly exhibited superior efficacy in treating cancer cells compared to PBS and Fe3O4. Furthermore, when an external magnetic field Fe-ATP(mag) was applied to the Fe-ATP self-assembly, it was confirmed that the Fe-ATP self-assembly was magnetically targeted, resulting in a higher efficacy in treating cancer cells. It was confirmed that the magnetically targeted Fe-ATP self-assembly inhibited cancer development to a level comparable to that of doxorubicin (DOX), a commonly used anticancer drug. On the other hand, it was confirmed that when only Fe3O4 was used, cancer development could not be inhibited due to low in vivo degradation efficiency, regardless of magnetic targeting.

[0230] Figure 11 demonstrated that magnetically targeted Fe-ATP self-assemblies effectively treat osteosarcoma cancer similarly to doxorubicin. In Figure 11, a high number of nuclei appearing purple in H&E staining indicates the presence of many cancer cells, while a high number of DNA fragments appearing brown in TUNEL staining indicates that a large number of cancer cells have been killed. Additionally, cancer tissues were represented by H&E staining using hematoxylin and eosin, as well as TUNEL staining. Immunohistochemistry (IHC) staining was performed, and in cancer cell death induced by Fe-ATP self-assemblies, the released LPO, GPX4, and Xc receptors were Fe released from the Fe-ATP self-assemblies. 2+ It was confirmed that it contributes to ferrotosis stimulated by the Fenton response.

[0231] Figure 12 shows the results of confirming the toxicity after implanting the Fe-ATP self-assembly into vivo. In Figure 12, it was confirmed that even after implanting the Fe-ATP self-assembly under a magnetic field in an osteosarcoma mouse model, the control group showed the same results as PBS, and no toxicity was observed in major organs in vivo. That is, the Fe-ATP self-assembly according to the present embodiment self-decomposes, and Fe 2+ It was confirmed that there is no toxicity in the human body even when emitted, and there is no toxicity even when a magnetic field is applied.

[0233] Figures 13 to 22 below show the experimental results related to Experimental Example 2.

[0235] Figure 13 shows the results confirming the chondroprotective effect of magnetically targeted Fe-ATP self-assemblies. Figure 13 is for Experimental Example 2, in which magnetically targeted Fe-ATP self-assemblies self-decompose to release ATP, and the released ATP induces polarization of anti-inflammatory macrophages, indicating that it is effective in treating osteoarthritis.

[0236] Figure 14 is a graph showing the cumulative profile of ATP release from the Fe-ATP self-assembly measured by HPLC. Before confirming the effect of ATP released from the Fe-ATP self-assembly in Experimental Example 2, ATP from the Fe-ATP self-assembly was used with PBS, which is similar to a biological fluid. The amount of release was verified. It was confirmed that the Fe-ATP self-assembly according to the present embodiment self-decomposes, and as previously examined, iron ions are released along with ATP. In addition, it was confirmed that approximately 30% of ATP is released from the Fe-ATP self-assembly over a period of about 25 days. The amount of ATP released from the Fe-ATP self-assembly according to the present embodiment can be varied by designing the Fe-ATP self-assembly in various ways.

[0237] Figures 15 to 17 show the experiments performed under each condition in cells prior to the experiment in rats according to Experimental Example 2 below. As shown in Table 6 below, each condition was classified into the control group (PBS only), Fe-ATP treatment, ATP only treatment, FeCl2 treatment, and Fe3O4 treatment.

[0239] division particular PBS (No treatment) Welgene, LB 204-02 Fe-ATP Synthesized Fe-ATP self-assembly (600 nm) ATP Daejeong Reagent, CAS: 51963-61-2 FeCl2 Iron(II) chloride, Sigma-Aldrich, cat. No. 372870 Fe3O4 Preparation Example 5 (10 nm)

[0241] Figure 15 shows the results of confirming the degradation of Fe-ATP self-assemblies. Referring to Figure 15, immunofluorescence staining of Arg-1 (M2 polarization marker), iNOS (M1 polarization marker), and Dapi was performed according to each condition. Arg-1 (M2 polarization marker) was not observed in PBS (no treatment), FeCl2, and Fe3O4, while it was confirmed that the M2 polarization marker Arg-1 was strongly observed only in samples containing ATP, specifically Fe-ATP self-assemblies and ATP. It was confirmed that Fe-ATP self-assemblies degrade to release ATP, which enables ATP-mediated anti-inflammatory polarization of macrophages for the treatment of osteoarthritis.

[0242] Figure 16 shows the results of confirming the polarization of macrophages by the degradation of Fe-ATP self-assemblies. Referring to Figure 16, it was confirmed that Fe-ATP self-assemblies self-degraded to release ATP, enabling ATP-mediated anti-inflammatory polarization of macrophages for the treatment of osteoarthritis, and this was confirmed through flow cytometry.

[0243] Figure 17 shows the results of confirming the degradation of the Fe-ATP self-assembly by western blotting. It was confirmed that the Fe-ATP self-assembly was degraded, enabling AIP-mediated anti-inflammatory polarization of macrophages. It was confirmed that low concentrations of ATP stimulated P2Y1 signaling, which promotes M2 polarization of macrophages, and low concentrations of ATP inhibited P2X7 signaling, which inhibits M2 polarization of macrophages.

[0244] Figure 18 shows C-Arm and 3-D micro CT images of the knee joint to confirm the therapeutic effect of Fe-ATP self-assemblies on osteoarthritis. C-Arm images of the knee joint were examined to identify ACLT and peripheral fractures in the osteoarthritis model at 0, 2, 4, 6, and 8 weeks. 3-D micro CT images were examined to identify ACLT and peripheral fractures in the osteoarthritis model at week 8. It was confirmed that Fe-ATP self-assemblies and magnetically targeted Fe-ATP self-assemblies were effective in treating osteoarthritis compared to PBS (no treatment).

[0245] Figure 19 shows 2-D micro CT images, H&E staining, and safranin-o(SO) staining images of a knee joint of an osteoarthritis model at week 8. Figure 19 confirms ACLT and peripheral fractures in the case treated with the Fe-ATP self-assembly according to the present embodiment. It was confirmed that the Fe-ATP self-assembly and magnetically targeted Fe-ATP self-assembly protect the cartilage of the knee joint, thereby preventing damage to the cartilage.

[0246] Figure 20 shows H&E, safranin-o(SO), and Collagen X staining images of the knee joint of an 8-week osteoarthritis model. In Figure 20, ACLT and peripheral fractures were observed in the 8-week osteoarthritis model. Fe-ATP autoassemblies and magnetically targeted Fe-ATP autoassemblies alleviated inflammation through anti-inflammatory macrophage polarization and inhibited the formation of hypertrophic differentiated chondrocytes, which mediate cartilage-to-bone transition. In particular, magnetically targeted Fe-ATP autoassemblies showed a higher effect than Fe-ATP autoassemblies. Here, Collagen X stained hypertrophic differentiated chondrocytes, confirming that hypertrophic differentiated chondrocytes promote cartilage-to-bone transition and exacerbate osteoarthritis.

[0247] Figure 21 shows iNOS and Arg-1 staining images of the synovial membrane of the knee joint of an osteoarthritis model at week 8. In Figure 21, ACLT and peripheral fractures of the osteoarthritis model were identified. It was confirmed that Fe-ATP self-assemblies protect the knee joint from cartilage degradation by promoting M2 polarization of macrophages through P2Y1 signaling, and that magnetically targeted Fe-ATP self-assemblies are particularly more effective.

[0248] Figure 22 shows the toxicity results of the Fe-ATP self-assembly confirmed in an osteoarthritis rat model. The results were confirmed after implanting the Fe-ATP self-assembly into various sites under a magnetic field, and no in vivo toxicity was observed in major organs. In other words, it was confirmed that the Fe-ATP self-assembly according to the present embodiment is not toxic to the human body even when it self-decomposes to release ATP, and is not toxic even when a magnetic field is applied.

[0250] Figures 23 to 32 below show the experimental results related to Experimental Example 3.

[0252] Figure 23 is a schematic diagram showing the bone regeneration effect of a 3D Fe-AMP self-assembly.

[0253] Figure 23 relates to Experimental Example 3, and it can be confirmed that the 3D Fe-AMP self-assembly exhibits a bone regeneration effect through the differentiation of stem cells into osteoblasts mediated by adenosine-A2B receptor signaling. The 3D Fe-AMP self-assembly was formed as a three-dimensional macroporous aggregate to enable the recruitment of self-movement-based cells for degraded-AMP-mediated fracture treatment through adenosine-A2B receptor signaling.

[0254] FIG. 24 is an SEM image showing a micropore structure in which a 3D Fe-AMP self-assembly is aggregated. The Fe-AMP self-assembly according to the present embodiment can be manufactured in a 3D form having a micropore structure, and its size can be varied from several micrometers to tens of centimeters. In addition, the Fe-AMP self-assembly manufactured in this manner can self-degrade in vitro or in vivo, and upon self-degradation, the iron ion Fe 2+ Or it was confirmed that AMP was emitted.

[0255] FIG. 25 is a diagram showing the magnetic targeting of a 3D-shaped Fe-AMP self-assembly. Referring to FIG. 25, when an external magnetic field is applied to the 3D-shaped Fe-AMP self-assembly, the movement of the 3D-shaped Fe-AMP self-assembly could also be controlled.

[0256] Figure 26 is a graph showing the AMP released over time from the Fe-AMP self-assembly. Before confirming the effect of the AMP released from the Fe-AMP self-assembly in Experimental Example 3, the amount of AMP released from the Fe-AMP self-assembly was confirmed using PBS, which is similar to a biological fluid. It was confirmed that the Fe-AMP self-assembly according to the present embodiment self-decomposes and releases AMP over time, and the release time lasts for approximately 25 days, with approximately 40% being released. The amount of AMP released from the Fe-AMP self-assembly according to the present embodiment can be varied by designing the Fe-AMP self-assembly in various ways.

[0257] Figures 27 and 28 show the experiments performed under each condition in cells prior to experimentation in rabbits according to Experimental Example 3 below. As shown in Table 7 below, each condition was classified into the control group (PBS only), Fe-AMP treatment, AMP only treatment, combined treatment of Adenosine and PO4 (Adenosine + PO4), Adenosine treatment, PO4 treatment, FeCl2 treatment, and Osteogenic induction medium treatment. Here, in the case of FeCl2, it dissolves and Fe 2+ and Cl - Since it is broken down into, the Fe released from Fe-ATP 2+ It was added for comparison with.

[0259] division particular PBS (No treatment) Welgene, LB 204-02 Fe-AMP Synthesized Fe-AMP self-assembly (Preparation Example 4) AMP Alfa Aesar, CAS: 4578-31-8 Adenosine+PO4 대정시약, CAS: 6613-4405(Adenosine), Sigma, A9251(PO4) 1:1 mix Adenosine Sigma, A9251 PO4 대정시약, CAS: 6613-4405 FeCl2 Iron(II) chloride, Sigma-Aldrich, cat. No. 372870 Osteogenic induction medium DMEM (Gibco 11965-118), 10mM β-glycerophosphate (sigma G9422), 50μM ascorbic acid-2-phosphate (sigma A8960), 100 nM dexamethasone (sigma D2915) 4-substance mixture

[0261] Figure 27 shows the results confirming that osteogenic differentiation is promoted by AMP-degraded adenosine released from 3D Fe-AMP self-assemblies. The AMP-degraded adenosine released from Fe-AMP self-assemblies is similar to the osteogenic differentiation induction medium and was analyzed by immunofluorescence staining of RUNX2 and osteocalcin, along with actin, dapi-positive nuclei, and ALP chemostaining. Fe-AMP self-assemblies induced osteogenic differentiation of stem cells to an extent corresponding to osteogenic induction media that promotes osteogenic differentiation, and adenosine induced osteogenic differentiation to a similar degree. In other words, Fe-AMP self-assemblies induced osteogenic differentiation of stem cells through adenosine signaling, which could be confirmed by immunofluorescence staining of RUNX2 and osteocalcin along with actin, dapi-positive nuclei, and ALP chemical staining.

[0262] Figure 28 shows the Western blotting results confirming osteogenic differentiation by CD73-mediated AMP-degrading adenosine released from 3D Fe-AMP self-assemblies. Referring to Figure 28, CD73-mediated AMP-degrading adenosine released from Fe-AMP self-assemblies promotes osteogenic differentiation through adenosine-A2B receptor signaling, and the addition of an A2B receptor inhibitor (PSB 603) inhibited adenosine-A2B receptor signaling, thereby inhibiting osteogenic differentiation of hMSCs. It was confirmed that CD73 degrades AMP into adenosine+PO4 and promotes osteogenic differentiation through adenosine-A2B receptor signaling.

[0263] Figure 29 shows C-Arm images of the femur to confirm fractures at 0, 2, 4, 6, and 8 weeks. Referring to Figure 29, it was confirmed that the 3D Fe-AMP self-assembly of the present invention exhibited effects similar to Ca-phosphate, a commonly used fracture graft material, for the control group. Specifically, while the Short Fe-AMP self-assembly was effective in treating fractures compared to the control group, the magnetically targeted Short Fe-AMP(mag) self-assembly was more effective in treating fractures than the Short Fe-AMP self-assembly. Furthermore, it was confirmed that the Short Fe-AMP(mag) self-assembly and the Long Fe-AMP self-assembly treated fractures similarly to Ca-phosphate. In other words, when comparing the Short Fe-AMP self-assembly and the Short Fe-AMP(mag) self-assembly, it was confirmed that the Short Fe-AMP(mag) self-assembly can be moved by a magnet, thereby being more effective in treating fractures.

[0264] Figure 30 is a micro CT image of the femur for identifying fractures at 8 weeks. In Figure 30, the Short Fe-AMP(mag) autoassembly exhibited overall fracture healing attributed to cell recruitment based on magnetic migration and reduced AMP-mediated adenosine-A2B receptor signaling. The Long Fe-AMP autoassembly exhibited overall fracture healing, which was confirmed to be attributed to reduced AMP-mediated adenosine-A2B receptor signaling. The effects of fracture healing were observed in the order of Long Fe-AMP autoassembly, Short Fe-AMP(mag) autoassembly, and Short Fe-AMP autoassembly, with the Long Fe-AMP autoassembly showing an effect almost similar to that of Ca-phosphate. In addition, in Figure 30, a lower P value indicates a larger difference between the two values, and the values ​​were found to be p=0.074 for Control vs Short Fe-AMP, p<0.001 for Control vs Short Fe-AMP(mg), p<0.001 for Control vs Long Fe-AMP, p<0.001 for Control vs Ca-phosphate, and P=0.008 for Short Fe-AMP vs Short Fe-AMP(mg).

[0265] Figure 31 shows H&E, osteocalcin, and TRAP stained images of the fracture. Short Fe-AMP(mag) autoassemblies exhibited overall fracture healing attributed to cell recruitment based on magnetic migration and reduced AMP-mediated adenosine-A2B receptor signaling. Long Fe-AMP autoassemblies exhibited overall fracture healing, which was confirmed to be attributed to reduced AMP-mediated adenosine-A2B receptor signaling.

[0266] Referring to Figure 31, the morphology of the tissue was confirmed through H&E staining images, the degree of osteogenic differentiation through osteocalcin staining images, and the presence or absence of osteoclasts through TRAP staining images. In the Long Fe-AMP self-assemblies and Short Fe-AMP(mag) self-assemblies, it was confirmed that bone was formed by connecting up to the center of the defect; judging by the activation of osteoclasts, it appears that the breakdown and regeneration of the bone environment were promoted, creating an environment for bone growth. In the case of the Short Fe-AMP self-assemblies, it was confirmed that the newly formed bone could not connect all the defect sites because no magnetic field was applied.

[0267] Figure 32 shows the results of confirming the toxicity of the in vivo 3D Fe-AMP self-assembly. After implanting the 3D Fe-AMP self-assembly into a fracture-healing rabbit model, it was examined under a magnetic field, and it was confirmed that no toxicity was observed in major organs.

[0269] As described above, the Fe-ATP self-assembly or Fe-AMP self-assembly according to the present embodiment is non-toxic in vivo and can self-decompose in vivo or in vitro to release iron ions or ATP / ADP, and the amount and rate of release can be controlled. In addition, since it is paramagnetic, it can be magnetically targeted by controlling its movement to a specific location upon the application of an external magnetic field. Furthermore, no toxicity was found in vivo even when the Fe-ATP self-assembly or Fe-AMP self-assembly was magnetically targeted.

[0270] Specifically, Fe-ATP self-assemblies are effective in treating cancer by inducing cancer cell death through ferrotosis. Additionally, Fe-ATP self-assemblies can treat osteoarthritis by releasing ATP, and Fe-AMP self-assemblies can act as implants for bone defects.

[0272] A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without altering its technical concept or essential features. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention is defined by the claims set forth below rather than by the detailed description above, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included within the scope of the present invention.

Claims

Claim 1 A therapeutic magnetic biomolecule-metal ion self-assembly complex comprising: an iron ion; and a ligand which is one or more of AMP (adenosine monophosphate) and ATP (adenosine triphosphate); wherein the ligand and the iron ion reversibly self-assemble or self-decompose, and the ligand and the iron ion self-assemble by means of a first bond including a coordination bond to form a self-assembly body, wherein the self-assembly body is composed of the ligand and the iron ion which are biomolecules, and wherein the self-assembly body is provided in a plurality of such units, and adjacent self-assemblies are self-bonded by means of a second bond including one or more of hydrogen bonds and π-π interactions, wherein the self-assembly body has paramagnetism due to the self-assembly of the iron ion and the ligand, and its movement is controlled by the application of an external magnetic field, and wherein the self-assembly body self-decomposes to release one or more of the iron ion and the ligand, but does not exhibit toxicity in normal cells. Claim 2 A therapeutic magnetic biomolecular-metal ion self-assembly complex according to claim 1, wherein the self-assemblies or adjacent self-assemblies are self-assembled for a first time period or self-decomposed for a second time period under physiologically relevant conditions, wherein the first time period is 1 minute to 24 hours and the second time period is 1 day to 90 days. Claim 3 A therapeutic magnetic biomolecular-metal ion self-assembly complex according to claim 1, wherein the self-assembly promotes self-decomposition under at least one of conditions including a chelating agent, strong acid conditions, and strong base conditions, wherein under conditions including a chelating agent, the chelating agent is one or more of EDTA (ethylenediaminetetraacetic acid), bipyridyl, and ferrozine, the pH of the strong acid conditions is 2 to 5, and the pH of the strong base conditions is 9 to 12. Claim 4 A therapeutic magnetic biomolecular-metal ion self-assembly complex according to claim 1, wherein when the ligand is ATP (adenosine triphosphate), the therapeutic magnetic biomolecular-metal ion self-assembly complex is provided as individual spherical shapes, with the average diameter of the self-assemblies being 70 nm to 600 nm, and when the ligand is AMP (adenosine monophosphate), the therapeutic magnetic biomolecular-metal ion self-assembly complex is provided as a three-dimensional aggregate having micropores formed by the aggregation of a plurality of self-assemblies. Claim 5 A therapeutic magnetic biomolecular-metal ion self-assembly complex according to claim 1, wherein the ligand is ATP (adenosine triphosphate), and the self-assembly unit releases the ATP during self-decomposition to promote M2 polarization of the macrophages through P2Y1 receptors of the macrophages distributed in the synovial membrane of the bone joint, and the M2 polarization of the macrophages maintains an anti-inflammatory environment of the synovial fluid of the bone joint to protect the osteochondral structure, thereby preventing, improving, or treating osteoarthritis. Claim 6 A therapeutic magnetic biomolecular-metal ion self-assembly complex according to claim 5, wherein the self-assembly moves to a target bone joint site upon the application of the external magnetic field to protect the osteochondral tissue and thereby prevent, improve, or treat osteoarthritis. Claim 7 A therapeutic magnetic biomolecular-metal ion self-assembly complex according to claim 5, wherein the ATP released from the self-assembly is controlled at a concentration of 5 to 10 μM in the synovial fluid of a bone joint to stimulate P2Y1 signaling that promotes M2 polarization of macrophages and inhibits P2X7 signaling that inhibits M2 polarization of macrophages. Claim 8 In claim 5, the above-mentioned self-assembled therapeutic magnetic biomolecular-metal ion self-assembly complex inhibits the formation of collagen X-positive hypertrophic differentiated chondrocytes in the cartilage of bone joints. Claim 9 A therapeutic magnetic biomolecular-metal ion self-assembly complex according to claim 1, wherein the ligand is AMP (adenosine monophosphate), and the therapeutic magnetic biomolecular-metal ion self-assembly complex is provided with a plurality of self-assemblies aggregated to form a three-dimensional aggregate having micropores, and the three-dimensional aggregate has an average diameter of 500 μm to 10 cm. Claim 10 A therapeutic magnetic biomolecular-metal ion self-assembly complex according to claim 9, wherein the three-dimensional aggregate is provided with a network structure in which the micropores are interconnected three-dimensionally. Claim 11 A therapeutic magnetic biomolecular-metal ion self-assembly complex according to claim 9, wherein the self-assembly is used as a bone graft material that restores the bone tissue by being implanted into a bone defect site in the form of a three-dimensional aggregate, host cells are attached to the surface of the self-assembly to form a skeleton, and the host cells attached to the surface of the self-assembly also move together upon the application of an external magnetic field. Claim 12 A therapeutic magnetic biomolecular-metal ion self-assembly complex according to claim 9, wherein the self-assembly is implanted into a bone defect site, the self-assembly self-decomposes to release the AMP for 1 to 70 days, the released AMP is decomposed into adenosine and phosphate by CD73 on the surface of a host cell to promote signaling of adenosine receptors on the surface of the host cell, the adenosine receptors include adenosine A2B receptors, and the signaling of the adenosine receptors promotes osteogenic differentiation of mesenchymal stem cells (MSCs).

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