Radiopharmaceutical using nanoparticles and method for producing same

Abstract

The present invention relates to a radiopharmaceutical using nanoparticles and a method for producing same. The present invention uses nanoparticles incorporating iodine bridges so that the nanoparticles can be selectively delivered to a tumor site, thereby improving the accuracy and efficiency of cancer treatments and minimizing various side effects caused by in vivo accumulation of detached isotopes.

Description

Radiopharmaceuticals using nanoparticles and their manufacturing methods

[0001] The present invention provides a radiopharmaceutical using nanoparticles and a method for manufacturing the same.

[0002] Cancer is a leading cause of death worldwide, and various cancer treatment methods are being studied. In particular, tumor-targeted therapy is attracting attention as a strategy that targets cancer cells specifically and minimizes damage to normal cells. HER2 (Human Epidermal Growth Factor Receptor 2) is a protein expressed in various cancers, including breast cancer, and HER2-targeted therapy plays a crucial role in cancer treatment. Trastuzumab, a monoclonal antibody targeting HER2, is widely used to treat HER2-positive breast cancer. However, trastuzumab monotherapy has limitations, such as drug resistance and incomplete removal of cancer cells.

[0003] Recently, nanotechnology-based cancer treatments are being actively researched. Nanoparticles, a technology that can enhance targeting and drug delivery efficiency, are being used in combination with various cancer treatments. In particular, nanoparticles loaded with radioactive isotopes are next-generation radiopharmaceuticals and have the potential to maximize the effectiveness of various intractable cancers that have previously been difficult to treat.

[0004] Existing radiopharmaceuticals have limitations, including poor labeling efficiency, which reduces drug delivery efficiency. Furthermore, the desorbed radioisotope can accumulate in normal tissues, such as bone marrow and kidneys, reducing its therapeutic efficacy against various cancers. To overcome these limitations, efforts have been made to increase drug doses, use less toxic radioisotopes, and develop targeting ligands. However, significant limitations remain in their application to various refractory cancers, necessitating continued research.

[0005] The purpose of the present invention is to provide a nanoparticle and a method for producing the same, characterized in that it comprises an inorganic core formed by a lattice structure compound interface layer containing a radioactive isotope, and the interface layer is further coated with an inorganic shell or organic shell to which a targeting ligand is covalently bonded.

[0006] Another object of the present invention is to provide a pharmaceutical composition for treating or preventing cancer, which comprises the nanoparticles as an active ingredient.

[0007] The present invention aims to improve the selective treatment effect for various intractable cancers and minimize side effects by maintaining the target specificity of existing antibody therapy while providing an additional cell killing effect using a radioactive isotope.

[0008] The present invention utilizes nanoparticles with an iodine bridge to overcome the problem of existing antibodies accumulating in the liver, lungs, bone marrow, and kidneys, thereby facilitating the excretion of radioactive isotopes or nanoparticles from the body after treatment.

[0009] The present invention provides a multifunctional treatment system that can overcome the limitations of existing radiopharmaceuticals by combining various targeting ligands such as antibodies, peptides, and small molecules with nanoparticles that have enhanced labeling efficiency through minimization of radioisotope desorption, and enables concurrent treatment with various chemical or immunotherapies, thereby improving the treatment efficiency of various intractable cancers.

[0010] In order to achieve the above object, the present invention provides a nanoparticle characterized in that it comprises an inorganic core formed with a lattice structure compound interface layer containing a radioactive isotope, and the interface layer is further coated with an inorganic shell or organic shell to which a targeting ligand is covalently bonded, wherein the lattice structure compound is M x X y, wherein M is copper (Cu), silver (Ag) or lutetium (Lu), X is one of fluorine (F), chlorine (Cl), bromine (Br) and iodine (I), x is one of the integers from 1 to 6, y is one of the integers from 1 to 6, and the inorganic shell or organic shell may further include iodine oxide to form an iodine bridge.

[0011] In addition, the present invention provides a pharmaceutical composition for treating or preventing cancer, which comprises the nanoparticles as an active ingredient.

[0012] The present invention provides the effect of selectively accumulating radioactive nanoparticles in HER2-expressing cancer cells, enabling smooth discharge, increasing the effectiveness of anticancer treatment, and reducing side effects caused by drug accumulation in the body, by chemically bonding nanoparticles with an iodine bridge that can minimize in vivo desorption of radioactive isotopes, and provides the effect of eliminating cancer cells resistant to various chemical drugs through concurrent radioisotope treatment, which solves the problem that drug resistance may occur in some cancer cells that occur in chemotherapy or immunotherapy alone.

[0013] Figure 1 is a drawing for explaining the configuration of nanoparticles according to the present invention.

[0014] FIG. 2 is a drawing showing a radioactive isotope included in a lattice structure of a metal oxide according to one embodiment of the present invention.

[0015] Figure 3 is a drawing visually comparing the effect of daughter nuclei generated during the nuclear decay process of a radioactive isotope on a conventional radiopharmaceutical and the effect on a radiopharmaceutical according to one embodiment of the present invention.

[0016] Figure 4 is a diagram schematically illustrating a method for manufacturing a radiopharmaceutical using nanoparticles according to one embodiment of the present invention.

[0017] Figures 5a and 5b are drawings comparing a one-step coating method and a multi-step coating method.

[0018] Figures 6 and 7 are a manufacturing method according to one embodiment of the present invention, metal oxide and M x X y A drawing showing an example of a step of combining a radioactive isotope into a weapon core including a radioactive isotope and coating the surface of the weapon core, separated into two steps.

[0019] FIG. 8A is a diagram illustrating a single-step reaction for binding a targeting ligand to a CM (Carboxymethyl)-dextran-coated inorganic core according to some embodiments of the present invention.

[0020] FIG. 8b is a diagram illustrating a multi-step reaction for binding a target ligand to a CM (Carboxymethyl)-dextran-coated inorganic core according to some embodiments of the present invention.

[0021] FIG. 9 is a diagram showing a reaction of binding a target ligand using Protein A to an inorganic core coated with CM (Carboxymethyl)-dextran according to some embodiments of the present invention.

[0022] FIG. 10 is a drawing for explaining an example of attaching an antibody to a nanoparticle according to one embodiment of the present invention.

[0023] Figure 11 is a schematic diagram showing an antibody functionalization step according to one embodiment of the present invention.

[0024] FIG. 12 is a diagram illustrating steps for producing a nanoparticle-antibody binding complex according to another embodiment of the present invention.

[0025] Figure 13 is a schematic diagram showing an antibody functionalization step of attaching a thiol functional group to an antibody according to one embodiment of the present invention.

[0026] FIG. 14 is a drawing showing a step of generating a nanoparticle-antibody binding complex using an antibody containing a thiol group (-SH) according to one embodiment of the present invention.

[0027] FIG. 15 is a diagram illustrating a step of generating an antibody chemically bound to a linker molecule using a linker molecule including an NHS ester according to one embodiment of the present invention.

[0028] FIG. 16 is a drawing showing a step of generating a nanoparticle-antibody binding complex using an antibody containing tetrazine according to one embodiment of the present invention.

[0029] FIG. 17 is a diagram showing the results of size analysis (DLS) of an anti-Her2 nanoparticle conjugate according to one embodiment of the present invention.

[0030] FIG. 18 is a drawing showing the analysis results of an Anti-Her2-nanoparticle conjugate complex using Cryo-TEM (cryo-electron microscopy) according to one embodiment of the present invention.

[0031] Figure 19 is a drawing showing the results of an anti-Her2 antibody binding analysis using ELISA according to one embodiment of the present invention.

[0032] Figure 20 is a drawing showing the results of evaluating the serum stability (plasma stability) of a nanoparticle-antibody binding complex according to one embodiment of the present invention in various biological species (human, mouse, rat, beagle).

[0033] FIG. 21 is a drawing showing an image taken using a fluorescence microscope to confirm cellular uptake of antibody-conjugated nanoparticles (Lu177-IONP-Trastuzumab-Fluor545) in breast cancer cells (BT-474 and MDA-MB-231) according to one embodiment of the present invention.

[0034] FIG. 22 is a drawing showing the results of comparing the degree of intracellular uptake of antibody-conjugated nanoparticles (Lu177-IONP-Trastuzumab-Fluor545) and antibody-free nanoparticles (Lu177-IONP-Fluor545) in BT-474 (HER2 positive cell line) and MDA-MB-231 (HER2 low-expressing cell line) according to one embodiment of the present invention.

[0035] FIG. 23 is a diagram showing the results of an In vitroEfficacy Evaluation experiment comparing the anticancer effects of antibody-conjugated nanoparticles (Lu177-IONP-Trastuzumab) and antibody-free nanoparticles (Lu177-IONP) according to one embodiment of the present invention on breast cancer cells (BT-474 and MDA-MB-231).

[0036] Figure 24 is a drawing showing the results of an experiment to confirm the biodistribution of antibody-conjugated nanoparticles (Lu177-IONP-Trastuzumab) according to one embodiment of the present invention.

[0037] FIG. 25 is a diagram showing the results of a serum biochemical analysis performed to evaluate liver function and kidney function in an experimental animal (mouse) administered antibody-conjugated nanoparticles (Lu177-IONP-Trastuzumab) according to one embodiment of the present invention.

[0038] Figure 26 is a drawing showing the results of an experiment to confirm the in vivo anticancer efficacy of antibody-conjugated nanoparticles (Lu177-IONP-Trastuzumab) according to one embodiment of the present invention.

[0039] Figure 27 shows the extent of accumulation in the body depending on whether or not iodine oxide was used in the lipid nanoparticle shell.

[0040] Hereinafter, the present invention will be described in more detail.

[0041]

[0042] The present invention provides a nanoparticle characterized in that it comprises an inorganic core formed by a lattice structure compound interfacial layer containing a radioactive isotope, and the interfacial layer is further coated with an inorganic shell or organic shell to which a targeting ligand is covalently bonded, wherein the lattice structure compound is M x X y , wherein M is copper (Cu), silver (Ag) or lutetium (Lu), X is one of fluorine (F), chlorine (Cl), bromine (Br) and iodine (I), x is one of the integers from 1 to 6, y is one of the integers from 1 to 6, and the inorganic shell or organic shell may further include iodine oxide to form an iodine bridge.

[0043] The above radioactive isotopes are iodine (I)-123, iodine (I)-125, iodine (I)-131, gallium (Ga)-67, gallium (Ga)-68, copper (Cu)-64, copper (Cu)-67, gold (Au)-198, lead (Pb)-210, nickel (Ni)-63, dysprosium (Dy)-165, radium (Ra)-226, lanthanum (La)-140, rhenium (Re)-186, rhenium (Re)-188, ruthenium (Ru)-82, lutetium (Lu)-177, manganese (Mn)-54, molybdenum (Mo)-99, bismuth (Bi)-213, samarium (Sm)-153, cesium (Ce)-137, Sodium (Na)-24, Scandium (Sc)-46, Strontium (Sr)-82, Strontium (Sr)-85, Strontium (Sr)-89, Strontium (Sr)-90, Americium (Am)-241, Zinc (Zn)-65, Erbium (Er)-169, Uranium (U)-234, Uranium (U)-235, Uranium (U)-238, Silver (Ag)-110m, Iridium (Ir)-192, Iridium (Ir)-169, Iridium (Ir)-177, Yttrium (Yt)-169, Yttrium (Yt)-177, Indium (In)-111, Germanium (Ge)-68, Iron (Fe)-55, It can be any one of cadmium (Cd)-109, calcium (Ca)-47, californium (Cf)-252, cobalt (Co)-57, cobalt (Co)-60, curium (Cm)-244, chromium (Cr)-51, chromium (Cr)-57, krypton (Kr)-81, krypton (Kr)-85, thallium (Tl)-201, thallium (Tl)-204, technetium (Tc)-99m, thorium (Th)-229, thorium (Th)-230, lead (Pd)-103, potassium (K)-42, polonium (Po)-210, promethium (Pm)-147, plutonium (Pu)-238, actinium (Ac)-225 and radium (Ra)-223, but It is not limited.

[0044] The above lattice structure compound can combine with a radioisotope to form a radioisotope halide bridge.

[0045] The above-mentioned inorganic core contains a metal oxide, and the metal of the metal oxide may be one or more of iron (Fe), scandium (Sc), titanium (Ti), vanadium (V), chromium (Cr), manganese (Mn), cobalt (Co), nickel (Ni), copper (Cu), yttrium (Y), zirconium (Zr), niobium (Nb), molybdenum (Mo), technetium (Tc), ruthenium (Ru), rhodium (Rh), palladium (Pd), silver (Ag), hafnium (Hf), tantalum (Ta), tungsten (W), rhenium (Re), osmium (Os), iridium (Ir), platinum (Pt), gold (Au), bismuth (Bi), and silicon (Si), but is not limited thereto.

[0046] The above metal oxide is Fe 13 O 19 , Fe3O4 (magnetite), γ-Fe2O3 (magemite) and α-Fe2O3 (hematite), β-Fe2O3 (beta phase), ε-Fe2O3 (epsilon phase), FeO (Wustite), FeO2 (Iron Dioxide), Fe4O5, Fe5O6, Fe5O7, Fe 25 O 32 and CuFeO2(Delafossite), but is not limited thereto.

[0047] The target ligand is at least one of a peptide, a small molecule, and an antibody, and the peptide is any one of somatostatin, GRP (Gastrin-releasing-peptide), GUL (Glu-Urea-Lys), and PSMA-I&T (Glu-Ureabased ligand), and the small molecule is fructose, levan, glucose, adenosine, glycine, glycine, tryptophane, alanine, arginine, lysine, riboflavin, biotin, thiamine, vitamin B12, methotrexate, anisamide, Anacardic acid, phenylboric acid, tamoxifen, folate, imatinib, vemurafenib, and sorafenib, wherein the antibody is a chimeric monoclonal antibody, a humanized monoclonal antibody, a human monoclonal antibody, an antibody fragment, an antibody-drug conjugate (ADC), an immunoconjugate, Trastuzumab, Pertuzumab, Ado-Trastuzumab Emtansine, Trastuzumab Deruxtecan, Rituximab, Cetuximab, Nivolumab, Bevacizumab,May be one of, but is not limited to, Atezolizumab, Blinatumomab, Olaratumab, Daratumumab, Elotuzumab, Ipilimumab.

[0048] The above-mentioned inorganic shell or organic shell is composed of an organic polymer and an inorganic polymer, respectively, and the organic polymer or the inorganic polymer may be any one of polyaniline, polyisobutylene, polyethylene, polystyrene, paraffin, graphene, polyvinylpyrrolidone, polyethylene glycol, gelatin, propylene glycol, poly(3,4-ethylenedioxythiophene), poly(methyl methacrylate), and polydimethylsiloxane, but is not limited thereto.

[0049] The above nanoparticles form a chemical bond with the radioisotope at the interface layer through a halogen element among the constituent elements of the halide compound, thereby preventing desorption of the radioisotope, and the iodine oxide can reduce specific or nonspecific binding with non-target substances other than target biomolecules.

[0050] The above nanoparticles may have a diameter (hydrodynamic size) of 1 nm or more and 200 nm or less.

[0051]

[0052] In addition, the present invention provides a pharmaceutical composition for treating or preventing cancer, which comprises the nanoparticles as an active ingredient.

[0053] The above cancer disease may be one of, but is not limited to, breast cancer, lung cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, colon cancer, brain tumor, glioblastoma, ovarian cancer, pancreatic cancer, stomach cancer, and esophageal cancer.

[0054] The above pharmaceutical composition may be a radiopharmaceutical for targeted therapy.

[0055] In another embodiment of the present invention, the pharmaceutical composition may further comprise one or more additives selected from the group consisting of suitable carriers, excipients, disintegrants, sweeteners, coating agents, bulking agents, lubricants, glidants, flavoring agents, antioxidants, buffers, bacteriostatic agents, diluents, dispersants, surfactants, binders and lubricants commonly used in the manufacture of pharmaceutical compositions.

[0056] Specifically, carriers, excipients, and diluents may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like. These solid preparations may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like, into the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents can be propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can include witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin.

[0057] According to one embodiment of the present invention, the pharmaceutical composition can be administered to a subject in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes.

[0058] The dosage of the active ingredient according to the present invention may vary depending on the condition and weight of the subject, the type and degree of the disease, the drug form, the route and period of administration, and may be appropriately selected by a person skilled in the art, and the daily dosage may be 0.01 mg / kg to 200 mg / kg, preferably 0.1 mg / kg to 200 mg / kg, and more preferably 0.1 mg / kg to 100 mg / kg. Administration may be once a day or divided into several times, and the scope of the present invention is not limited thereby.

[0059] Hereinafter, to aid understanding of the present invention, examples and other embodiments will be described in detail. However, the following examples and other embodiments merely illustrate the content of the present invention and are not intended to limit the scope of the present invention. The examples and other embodiments of the present invention are provided to more fully explain the present invention to those of average skill in the art.

[0060]

[0061] [Example] Overall configuration example

[0062] FIG. 1 is a drawing for explaining the composition of a nanoparticle according to the present invention, and discloses a nanoparticle (100) composition that stably contains a radioactive isotope (140) therein and prevents the radioactive isotope (140) from being randomly discharged from a normal organ in the body, and a method for producing the same.

[0063] The present invention discloses a nanoparticle (100) composition and a method for manufacturing the same, wherein an inorganic core (120) is coated with a flexible organic shell or inorganic shell (160) to secure an appropriate blood circulation time of a radiopharmaceutical and induce smooth renal excretion, thereby maximizing drug efficacy while minimizing renal toxicity.

[0064] The present invention discloses a nanoparticle (100) composition and a method for manufacturing the same, which stably contains a large amount of radioactive isotope (140) by introducing an iodine bridge (150), prevents side effects caused by the radioactive isotope being detached from an organ other than the target organ, and maximizes the drug effect in the target organ.

[0065] According to one embodiment of the present invention, a radioactive isotope (140) of lutetium-177 (Lu-177) or actinium-225 (Ac-225) was added together with NaI to an inorganic core (120) through an iodine bridge (150) to an inorganic nanocore into which CuF2 was introduced, and a microwave reaction was performed at 5 A, 200 W, 200°C for 5 minutes, preferably under vacuum conditions. Specifically, when CuF2 was added with NaI, polyethyleneglycol (PEG), and a radioactive isotope (140) of Lu-177 or Ac-225, and a microwave reaction was performed at 5 A, 200 W, 200°C for 5 minutes, preferably under vacuum conditions, it was confirmed that the F group of Cu was substituted with I. Accordingly, metallic radioactive isotopes can function as bridges at the interface of the inorganic core (120) by bridging between I and I.

[0066] A radiopharmaceutical utilizing nanoparticles (100) according to one embodiment of the present invention comprises a radioisotope (140), an inorganic core (120) including the radioisotope (140), a targeting ligand (180) attached to an organic shell or inorganic shell (160) surrounding the inorganic core (120), and an iodine bridge (150) for preventing desorption of the radioisotope (140) to organs other than the target organ.

[0067] In some embodiments, the inorganic core (120) including the radioisotope (140) includes that the inorganic core (120) and the radioisotope (140) are physically or chemically bonded or connected, which may be formed by forming a chemical bond through a coating and bridging process at the interface of the inorganic core (120), or by introducing a doping method such as a diffusion process or an ion implantation process.

[0068]

[0069] FIG. 2 is a drawing showing a radioactive isotope included in a lattice structure of a metal oxide according to one embodiment of the present invention. According to one embodiment of the present invention, an inorganic core (120) including a radioactive isotope (140) may further include a metal oxide. Meanwhile, the metal oxide includes a metal oxide capable of forming a lattice structure when agglomerated. The lattice structure of the metal oxide can semi-permanently include and / or capture the radioactive isotope (140) or semi-permanently bind to the radioactive isotope (140) to prevent arbitrary emission of the radioactive isotope (140).

[0070]

[0071] FIG. 3 is a drawing visually comparing the effect of daughter nuclei generated during the nuclear decay process of a radioisotope on a conventional radiopharmaceutical and the effect of daughter nuclei generated during the nuclear decay process of a radioisotope on a radiopharmaceutical according to an embodiment of the present invention. It was found that the lattice structure of the metal oxide according to an embodiment can play a role in stably including and / or capturing not only the radioisotope (140) itself present inside the nanoparticle (100) but also the daughter nuclei generated during the nuclear decay process of the radioisotope (140), thereby preventing damage to normal organs or the drug itself due to radiation emitted from the daughter nuclei.

[0072] The organic or inorganic shell (160) of a radiopharmaceutical utilizing nanoparticles (100) according to one embodiment of the present invention may further include a lipid nanoparticle shell. In this case, a structure in which the overall diameter of the radiopharmaceutical is within the range of 10 nm to 100 nm and the internal inorganic core diameter of the radiopharmaceutical is 2 nm to 10 nm is preferred. In one embodiment of the present invention, the overall diameter may be a hydrodynamic size.

[0073] Meanwhile, a structure can refer to any shape that has an internal, passable void space and an outer wall surrounding that void space. The diameter of the structure can refer to the diameter of the internal void space. Furthermore, the diameter of the internal void space can vary within a single structure. For example, a structure may include, but is not limited to, a human kidney drainage tract.

[0074] Meanwhile, the hydrodynamic diameter, which is one aspect of the total diameter, refers to the size of a virtual sphere that is assumed to diffuse in the same manner as the particle whose diameter is to be measured. For example, the hydrodynamic diameter, which is one aspect of the total diameter of a radiopharmaceutical utilizing a nanoparticle (100), may refer to the diameter of the particle that takes into account all of the inorganic core (120) of the nanoparticle (100), the organic shell (160) surrounding the inorganic core (120), and the targeting ligand (180) attached to the organic shell or the inorganic shell (160).

[0075] According to one embodiment of the present invention, even though the overall diameter of the nanoparticle (100) is 10 nm to 50 nm, which is larger than the diameter of the renal excretory passage (2 nm to 10 nm), the nanoparticle (100) can pass through a structure having a diameter in the range of 2 nm to 10 nm corresponding to the renal excretory passage.

[0076] According to one embodiment of the present invention, a radioisotope (140) and NaI may be placed between an inorganic core (120) and an organic polymer or an inorganic polymer, and a reaction may be performed for 5 minutes under conditions of 5 A, 200 W, and 200°C. In addition, a radioisotope (140) and NaI may be placed between an inorganic core (120) and an organic polymer or an inorganic polymer, and a microwave reaction may be performed under vacuum conditions to form a radioisotope interface layer. Specifically, in this process, some iodine ions may combine with oxygen of the organic polymer or oxygen of the inorganic polymer to be converted into hyper-valent iodine. Such hyper-valent iodine may minimize binding to macrophages and long-term accumulation, and may provide an effect of assisting excretion in the body.

[0077] According to one embodiment of the present invention, iodine present inside the organic shell or inorganic shell (160) of a radiopharmaceutical utilizing nanoparticles (100) may exist in the hypervalent form of iodoso or iodoxy by combining iodine and oxygen atoms in a ratio of 1:1 or more without following the octet rule, which is a basic characteristic of atoms. Such a form may exist by forming a bridge with the components of the organic shell or inorganic shell (160).

[0078] According to one embodiment of the present invention, a radiopharmaceutical utilizing nanoparticles (100) can block non-specific binding, protein binding, etc. in the body because it includes a hypervalent iodine bridge (150) inside an organic or inorganic shell (160). As a result, it can block the nanoparticles (100) from being fixed to a specific normal organ. In this case, the normal organ may be the liver. Accordingly, the present invention has the effect of preventing the side effect of a radiopharmaceutical being fixed to a normal organ and not being excreted, causing toxicity.

[0079] According to one embodiment of the present invention, the halide compound can be combined or linked with both the inorganic core (120) and the radioisotope (140). Specifically, the halide compound can serve to link the inorganic core (120) and the radioisotope (140). For example, the halide compound can be positioned between the inorganic core (120) and the radioisotope (140). If the inorganic core (120) further includes the halide compound during the manufacturing process, the radioisotope (140) can be included in the inorganic core (120) of the nanoparticle (100). In this manner, a large amount of the radioisotope (140) can be included within the nanoparticle (100). In this manner, the radiopharmaceutical can emit a large amount of radiation at the target organ, the radiotherapy effect can be maximized, and damage to normal cells can be minimized. According to one embodiment of the present invention, the halide compound may be, but is not limited to, CuI.

[0080] In some embodiments, the inclusion of a halide-based compound in the inorganic core (120) includes that the inorganic core (120) and the halide-based compound are physically or chemically bonded or connected. For example, the halide-based compound may be disposed within the inorganic core (120). For example, the inorganic core (120) and the halide-based compound may be bonded to each other through hydrogen bonding. For example, the halide-based compound may be coated on the surface of the inorganic core (120) through a general coating method. For example, the halide-based compound may be doped on the surface of the inorganic core (120) through a doping method such as a diffusion process or an ion implantation process. For example, the halide-based compound may form a layer and be coated on the surface of the inorganic core (120). However, the present invention is not limited thereto.

[0081] Specifically, when the halide compound is CuI, the I of CuI can bind to the inorganic core (120) in one direction and bind to the radioisotope (140) in the other direction. In this case, the radioisotope (140) added during the manufacturing process is included in the inorganic core (120) of the nanoparticle (100). Alternatively, for example, a radiopharmaceutical can be manufactured in which a large amount of the radioisotope (140) is included in the inorganic core (120) of the nanoparticle (100). In addition, the radiopharmaceutical can emit a large amount of radiation in the target organ.

[0082] According to one embodiment of the present invention, a halide-based compound is included in the inorganic core (120), and a radioisotope (140) is included in the inorganic core (120) of the nanoparticle (100) in a state of being physically or chemically bonded or connected to the halide-based compound. Therefore, the radioisotope (140) can be stably arranged in the inorganic core (120). For example, the radioisotope (140) can be coated on the interface of the inorganic core (120) in a state of being bonded to the halide-based compound. For example, the radioisotope (140) can be arranged inside the inorganic core (120) in a state of being bonded to the halide-based compound. For example, the radioisotope (140) can be coated on the interface of the inorganic core (120) in a state of being layered and bonded to the halide-based compound. However, the present invention is not limited thereto.

[0083]

[0084] -Example of nanoparticle manufacturing process

[0085] According to one embodiment of the present invention, a method for manufacturing a radiopharmaceutical using nanoparticles (100) includes: a step of mixing and heating a metal hydrate and an organic salt to form a hydrophobic organic compound-metal complex; a step of adding the complex to a halide-based compound and reacting the complex by heating to oxidize the metal and form an inorganic core (120) including a metal oxide and a halide-based compound; a step of mixing materials of an inorganic core (120), a radioisotope (140), and an organic shell (160) to bind at least one of the radioisotope (140) and M or X of the halide-based compound in the inorganic core (120), and coating the inorganic core (120) with the organic shell (160); and a step of binding a targeting ligand (180) to the organic shell (160). Hereinafter, a method for manufacturing a radiopharmaceutical using nanoparticles (100) will be described in detail. Figure 4 is a diagram schematically illustrating a method for manufacturing a radiopharmaceutical using nanoparticles according to one embodiment of the present invention.

[0086] 1) Step of preparing a hydrophobic organic compound-metal complex (S100)

[0087] According to one embodiment of the present invention, a method for manufacturing a radiopharmaceutical using nanoparticles (100) includes a step (S100) of preparing a hydrophobic organic compound-metal complex. Specifically, the step (S100) of preparing a hydrophobic organic compound-metal complex comprises preparing a hydrate of metal chloride (ACl n -xH2O) may refer to a step of converting a hydrophobic organic compound-metal complex.

[0088] According to one embodiment of the present invention, the step (S100) of preparing a hydrophobic organic compound-metal complex comprises adding a hydrophobic organic compound and a metal chloride hydrate (ACl) to a solvent. n-xH2O), a step of heating the mixture to react, a step of removing unreacted material through deionized water (DIW) washing, a step of heating and applying vacuum to remove the solvent, a step of obtaining a hydrophobic organic compound-metal complex, and a step of dissolving the complex in an oleyl alcohol / 1-octadecene solution and blending.

[0089] According to one embodiment of the present invention, a hydrate of a hydrophobic organic compound and a metal chloride (ACl n The solvent to which -xH2O is added may be, but is not limited to, ethanol / hexane.

[0090] The step of dissolving and blending the complex in an oleyl alcohol / 1-octadecene solution can improve the accuracy and convenience of quantitative measurement of a hydrophobic organic compound-metal complex in an oil form with viscosity and facilitate storage.

[0091] Meanwhile, the hydrophobic organic compound according to one embodiment of the present invention includes at least one compound selected from the group consisting of aliphatic hydrocarbon salts and amine compounds having 4 to 25 carbon atoms, but is not limited thereto.

[0092] According to one embodiment of the present invention, examples of the aliphatic hydrocarbon salt having 4 to 25 carbon atoms may include at least one selected from the group consisting of butyrate, valerate, caproate, enanthate, caprylic acid, pelargonate, caprate, laurate, myristate, pentadecyl acid, acetate, palmitate, palmitoleate, margarate, stearate, oleate, vaccenate, linoleate, (9,12,15)-linolenate, (6,9,12)-linolenate, eleostearate, tuberculostearate, rachidate, arachidonate, behenate, lignocerate, nervonate, ceroterate, montanate, melissate, and a peptide salt comprising one or more amino acids. These compounds may be used alone or in the form of a mixed salt of two or more. However, it is not limited to this.

[0093] According to one embodiment of the present invention, the metal component of the aliphatic hydrocarbon salt having 4 to 25 carbon atoms may include at least one selected from the group consisting of calcium (Ca), sodium (Na), potassium (K), and magnesium (Mg), but is not limited thereto.

[0094] According to one embodiment of the present invention, examples of amine compounds include methylamine, ethylamine, propylamine, isopropylamine, butylamine, amylamine, hexylamine, octylamine, 2-ethylhexylamine, nonylamine, decylamine, laurylamine, pentadecylamine, cetylamine, stearylamine and cyclohexylamine, dimethylamine, diethylamine, dipropylamine, diisopropylamine, dibutylamine, diamylamine, dioctylamine, di(2-ethylhexyl)amine, didecylamine, dilaurylamine, dicetylamine, distearylamine, methylstearylamine, ethylstearylamine and butylstearylamine, triethylamine, triamylamine, trihexylamine and trioctylamine, triallylamine and oleylamine, laurylaniline, stearylaniline, triphenylamine, N,N-dimethylaniline and A composition comprising at least one selected from the group consisting of dimethylbenzylaniline, monoethanolamine, diethanolamine, triethanolamine, dimethylaminoethanol, diethylenetriamine, triethylenetetramine, tetraethylenepentaamine, benzylamine, diethylaminopropylamine, xylylenediamine, ethylenediamine, hexamethylenediamine, dodecamethylenediamine, dimethylethylenediamine, triethylenediamine, guanidine, diphenylguanidine, N,N,N',N'-tetramethyl-1,3-butanediamine, N,N,N',N'-tetramethylethylenediamine, 2,4,6-tris(dimethylaminomethyl)phenol, morpholine, N-methylmorpholine, 2-ethyl-4-methylimidazole and 1,8-diazabicyclo(5,4,0)undecene-7(DBU). It may be, but is not limited to,

[0095] In one embodiment of the present invention, the metal in the hydrophobic organic compound-metal complex may include a metal that forms a certain lattice structure when aggregated in the form of an oxide. Specifically, the metal may include one or more metals selected from the group consisting of scandium (Sc), titanium (Ti), vanadium (V), chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), yttrium (Y), zirconium (Zr), niobium (Nb), molybdenum (Mo), technetium (Tc), ruthenium (Ru), rhodium (Rh), palladium (Pd), silver (Ag), hafnium (Hf), tantalum (Ta), tungsten (W), rhenium (Re), osmium (Os), iridium (Ir), platinum (Pt), and gold (Au), but is not limited thereto.

[0096] According to one embodiment of the present invention, the metal oxide can form a lattice structure upon aggregation. The lattice structure can stably contain and / or capture a radioisotope (140) that binds to the inorganic core (120) and prevent any release of the radioisotope (140).

[0097] According to one embodiment of the present invention, the metal oxide may include an impurity including silicon (Si) or copper (Cu). The impurity including silicon (Si) or copper (Cu) may serve to stabilize the surface of the nanoparticle (100).

[0098] According to one embodiment of the present invention, the metal oxide may be iron oxide in detail. The iron oxide according to one embodiment of the present invention is an oxide of iron, for example, Fe 13 O 19 , Fe3O4 (magnetite), γ-Fe2O3 (magemite) and α-Fe2O3 (hematite), β-Fe2O3 (betaphase), ε-Fe2O3 (epsilon phase), FeO (Wustite), FeO2 (IronDioxide), Fe4O5, Fe5O6, Fe5O7, Fe 25 O32 , may include at least one selected from the group consisting of ferrite type and delafossite, but is not limited thereto.

[0099]

[0100] 2) Step of preparing an inorganic core (120) containing a metal oxide and a halide compound (S200)

[0101] According to one embodiment of the present invention, a method for manufacturing a radiopharmaceutical using nanoparticles (100) includes a step (S200) of preparing an inorganic core (120) including a metal oxide and a halide compound.

[0102] According to one embodiment of the present invention, the step (S200) of preparing an inorganic core (120) including a metal oxide and a halide-based compound includes the steps of mixing the prepared hydrophobic organic compound-metal complex and the halide-based compound, heating and reacting the mixture to produce an inorganic core (120) including a metal oxide and a halide-based compound, performing centrifugation to collect an inorganic core (120) of a desired size, discarding the pellet obtained through centrifugation to remove a large-sized inorganic core (120), dispersing the desired inorganic core (120) through ultrasonic treatment, washing the dispersed inorganic core (120) with ethanol and hexane to remove impurities, dispersing the inorganic core (120) in hexane, filtering it through a syringe filter, and then removing the hexane through rotary evaporation, and measuring the weight of the dried inorganic core (120). The method may include adjusting the concentration of the suspension by adding toluene, and storing the suspension in a glass vial container covered with PTFE.

[0103] According to one embodiment of the present invention, in the step of generating an inorganic core (120) including a metal oxide and a halide compound by heating and reacting, the heating may be performed under nitrogen (N2) gas. For example, the heating may be performed under argon (Ar2) gas. For example, the heating may also be performed under a gas mixed with nitrogen (N2) and argon (Ar2). However, the present invention is not limited thereto.

[0104] According to one embodiment of the present invention, the halide compound may play a role in enabling the radioisotope (140) and the inorganic core (120) to be combined in the inorganic core (120) during the manufacturing process. In addition, the halide compound may play a role in controlling the size of the inorganic core (120).

[0105] According to one embodiment of the present invention, the halide compound, when added, can change the lattice shape of the lattice structure formed by the metal oxide contained within the inorganic core (120). Accordingly, the size of the inorganic core (120) can vary. Meanwhile, the size of the inorganic core (120) can be controlled by adjusting the ratio of the halide compound added during the manufacturing process.

[0106] According to one embodiment of the present invention, when the halide compound serves to enable the radioactive isotope (140) and the inorganic core (120) to be combined in the inorganic core (120) during the manufacturing process, the halide compound may be CuI.

[0107] According to one embodiment of the present invention, when a halide compound serves to control the size of the inorganic core (120) during the manufacturing process, the halide compound may be CuF2. The specific materials of the halide compound described above are merely examples for explanation and are not limited thereto.

[0108]

[0109] 3) Surface coating step (S300)

[0110] According to one embodiment of the present invention, a method for manufacturing a radiopharmaceutical using nanoparticles (100) includes a step (S300) of binding a radioactive isotope (140) to an inorganic core (120) including a metal oxide and a halide compound and coating the surface of the inorganic core (120).

[0111] According to one embodiment of the present invention, the step (S300) of binding a radioisotope (140) to an inorganic core (120) including a metal oxide and a halide compound and coating the surface of the inorganic core (120) may include the steps of adding a solution containing an organic shell (160) material including at least one organic polymer or peptide, a radioisotope (140), a target ligand (180), toluene, and ethanol to a solution including the inorganic core (120), stirring the reaction vessel to react, and then removing the solvent using a rotary evaporator, adding distilled water and then using an ultrasonic cleaner to disperse the inorganic core (120) to which the radioisotope (140) is bound and coated with the organic shell (160) and the target ligand (180), purifying the reaction solution through centrifugation or ion exchange chromatography, and exchanging and concentrating a buffer of the purified solution. (Hereinafter, single-step coating method)

[0112] In addition, according to one embodiment of the present invention, the step (S300) of combining a radioisotope (140) with an inorganic core (120) including a metal oxide and a halide compound and coating the surface of the inorganic core (120) includes the steps of: adding a solution containing an organic shell (160) material, a radioisotope (140), toluene, and ethanol, to a solution containing the inorganic core (120); stirring the reaction vessel to react; and then removing the solvent using a rotary evaporator; adding distilled water and then using an ultrasonic cleaner to disperse the inorganic core (120) to which the radioisotope (140) is combined and coated with the organic shell (160); separating the solution through a centrifuge; discarding the precipitate and taking the supernatant; adding a solution containing a target ligand (180) to the reaction vessel to coat the target ligand (180) with the organic shell. It may include a step of binding to the surface of the inorganic core (120), a second purification step of purifying the reaction solution through centrifugation or ion exchange chromatography, and a step of exchanging and concentrating the buffer of the purified solution. (Hereinafter, a multi-step coating method)

[0113] According to one embodiment of the present invention, the organic shell material may be combined with a water-soluble functionalized substance. The water-soluble functionalized substance may include a functional group that serves to introduce a target ligand (180) into the organic shell through a series of chemical reactions. Specifically, the water-soluble functionalized substance may include an amine group (-NH 2, amine group) or azide group (-N3 -, azide group) may be included, but is not limited thereto. Specifically, the organic shell (160) material may be combined with a water-soluble functionalized substance, which means that the organic shell (160) material and the water-soluble functionalized substance may be added together to the reaction solution, or the terminal of the compound included in the organic shell (160) material may include an amine group (-NH) derived from the water-soluble functionalized substance. 2, amine group) or azide group (-N3 - , azidegroup) may be substituted, but is not limited thereto.

[0114] Meanwhile, according to one embodiment of the present invention, a solution including a targeting ligand (180) may include a compound that can bind the targeting ligand (180) to the surface of the entire nanoparticle (100) including the organic shell (160) when added to the inorganic core (120) coated with the organic shell (160) by reacting with the organic shell (160) material in which the water-soluble functionalized substance is mixed. Specifically, the compound may be EDC (N-(3-Dimethylaminopropyl)-N'-ethylcarbodimide hydrochloride), Sulfo-NHS (N-Hydroxysuccinimide), targeting ligand (180)-DBCO (Dibenzocyclooctyne), maleimide, and targeting ligand (180)-azide group (-N3 - , azide group) complexes, but are not limited thereto.

[0115] According to one embodiment of the present invention, the water-soluble functionalized material comprises an amine group (-NH 2, In the case of a compound having an amine group, the outer terminal of the organic shell (160) surrounding the inorganic core (120) is an amine group (-NH 2,amine group). Here, when the target ligand (180) and EDC (N-(3-Dimethylaminopropyl)-N'-ethylcarbodimide hydrochloride) and Sulfo-NHS (N-Hydroxysuccinimide) are reacted together, the amine group (-NH) of the organic shell (160) is reacted through an amine-mediated EDC / NHS coupling reaction. 2, amine group) and the target ligand (180).

[0116] According to one embodiment of the present invention, the water-soluble functionalized material comprises an azide group (-N3 - , azide group), the outer terminal of the organic shell (160) surrounding the inorganic core (120) is an azide group (-N3 - , azide group). Here, when the target ligand (180)-DBCO (Dibenzocyclooctyne) complex is added and reacted, the azide group (-N3) of the organic shell (160) is formed through the azide-DBCO click chemistry technique. - , azide group) and a target ligand (180) can be combined.

[0117] According to one embodiment of the present invention, when the water-soluble functionalized material is a compound having DBCO (Dibenzocyclooctyne), the outer terminal of the organic shell (160) surrounding the inorganic core (120) can be substituted with DBCO (Dibenzocyclooctyne). Here, the target ligand (180) - azide group (-N3 - , azide group) complex is added to react, the DBCO (Dibenzocyclooctyne) of the organic shell (160) and the target ligand (180) can be combined through the azide-DBCO click chemistry technique.

[0118] According to the present invention, a one-step coating method and a multi-step coating method can be distinguished depending on whether the step of coating an inorganic core (120) with a hydrophilic organic shell (160) and the step of binding a targeting ligand (180) to the end of the organic shell (160) are performed in one reaction step or are performed separately in multiple reaction steps. In the one-step coating method, in the step of coating an inorganic core (120) with a hydrophilic organic shell (160), a targeting ligand (180) is also added to the reaction vessel to bind the targeting ligand (180) to the end of the organic shell (160) surrounding the inorganic core (120). In contrast, the multi-step coating method involves completing the step of coating the inorganic core (120) with a hydrophilic organic shell (160), then performing a first purification step, and then adding the target ligand (180) to the reaction vessel to bind the target ligand (180) to the end of the organic shell (160) surrounding the inorganic core (120).

[0119]

[0120] - Comparison of single-step and multi-step coating methods

[0121] As illustrated in FIGS. 5a and 5b, the multi-step coating method was able to produce nanoparticles (100) having a uniform distribution between the nanoparticles (100) and the targeting ligands (180) compared to the case of using the one-step coating method. In addition, it was confirmed that the multi-step coating method could increase the rate at which the targeting ligands (180) were activated after being bound to the surface of the inorganic core (120). In addition, according to one embodiment of the present invention, it was confirmed that the multi-step coating method could prevent the aggregation of the nanoparticles (100) by preventing the connection or binding between the targeting ligands (180) bound to the surface of the inorganic core (120).

[0122]

[0123] - Specific examples (entire manufacturing process)

[0124] According to one embodiment of the present invention, a method for manufacturing a radiopharmaceutical using a nanoparticle (100) comprising: an inorganic core (120) including iron oxide and CuF2; a radioisotope interface layer that is stabilized after ion-exchange from CuF2 to CuI; an organic shell (160) composed of polyethyleneglycol (PEG) surrounding the inorganic core (120); hyper-valent iodine connecting the organic shells (160); an antibody trastuzumab that is attached to the end of the organic shell (160) and functions as a targeting ligand (180); and a radioisotope (140) Lu-177 or Ac-225 that binds to iodine in the inorganic core (120) is described.

[0125] (1) Step of preparing a hydrophobic organic compound-metal complex (S100)

[0126] According to one embodiment of the present invention, the step (S100) of preparing a hydrophobic organic compound-metal complex is a step of converting iron (III) chloride hydrate (FeCl3-xH2O) into a hydrophobic iron oleate complex, and the main process is to mix iron (III) chloride hydrate (FeCl3-xH2O) with sodium oleate, ethanol, and hexane and heat to 110°C to cause a reaction.

[0127] 1) Initial synthesis: Sodium oleate, ethanol, hexane, and iron (III) chloride hydrate (FeCl3-xH2O) were mixed and heated at 110℃ for 9 hours to react, and the unreacted remaining hydrophilic substances were removed through washing (extraction) with deionized water (DI water).

[0128] 2) Dehydration of solution: The solution was heated at 150℃ for 12 hours to allow for reaction, and the solvent (nucleic acid) remaining after the reaction was removed by applying vacuum.

[0129] 3) Blending: Since the iron-oleate complex is in the form of a viscous oil, it was dissolved in an oleyl alcohol / 1-octadecene solution and blended in the mixing process to improve the accuracy and convenience of quantitative measurement and to facilitate storage.

[0130] (2) Metal oxide and M x X y Step (S200) of preparing a weapon core (120) including

[0131] 1) Synthesis: CuF2, an inorganic halide linker, was added to the hydrophobic iron oxide-oleate synthesized above, and heated under nitrogen (N2) gas to form an iron oxide inorganic core (120). CuF2 can serve as a size-controlling agent for inorganic nanocores and a precursor that stabilizes radioisotopes by being substituted with iodine. For example, CuF2 can function as a bridge at the interface that forms a stabilized layer of a radioisotope at the interface of the inorganic core (120). For another example, CuF2 can function as a dopant, and depending on the amount added, it can modify the lattice shape of the inorganic core (120) and control the size of the inorganic core.

[0132] 2) Centrifugation was performed to collect the inorganic core (120) of the desired size, the pellet was discarded to remove the large-sized inorganic core (120), and then the desired particles were dispersed through ultrasonic treatment.

[0133] 3) Cut off: The weapon core was washed with ethanol and hexane to remove impurities.

[0134] 4) Purification: The inorganic core (120) was dispersed in hexane, filtered through a syringe filter, and then the nucleic acid was removed through rotary evaporation.

[0135] 5) The weight of the dried inorganic core (120) was measured, toluene was added to adjust the suspension concentration, and the suspension was stored in a glass vial container covered with PTFE.

[0136]

[0137] (3) Metal oxides and M x X y A step of stabilizing a weapon core (120) and a radioactive isotope at the interface.

[0138] In the inorganic nanocore into which CuF2 was introduced, radioactive isotopes (140) Lu-177 or Ac-225 were added together with NaI in the inorganic core (120) through an iodine bridge (150), and microwave reaction was performed under conditions of 5 A, 200 W, 200 ° C., 5 minutes, and vacuum. Specifically, when CuF2 was added with NaI, polyethylene glycol (PEG), and radioactive isotopes (140) of Lu-177 or Ac-225, and microwave reaction was performed under conditions of 5 A, 200 W, 200 ° C., 5 minutes, preferably, and vacuum, the F group of Cu was replaced with I. Accordingly, it was confirmed that metallic radioactive isotopes can function as bridges at the interface of the inorganic core (120) by bridging between I and I.

[0139]

[0140] (4) Step (S300) of combining a radioactive isotope (140) with an inorganic core (120) including a metal oxide and a halide compound and coating a biocompatible organic shell or inorganic shell (160)

[0141] 1) Step of stabilizing the radioisotope (140) at the interface of the inorganic core (120): The radioisotope (140) was stabilized at the interface of the inorganic core (120) through an iodine bridge to the inorganic nanocore into which CuF2 was introduced. The inorganic core (120) and a radioisotope (140) such as Lu-177 or Ac-225 were added together with NaI, and a microwave reaction was performed at 5 A, 200 W, 200°C for 5 minutes, preferably under vacuum conditions. When the microwave reaction was performed, the F group of CuF2 of the inorganic core (120) was replaced with I. At this time, it was confirmed that I could function as a bridge that connects the radioisotope (140) at the interface of the inorganic core (120).

[0142] 2) Biocompatible organic or inorganic shell (160) coating: The surface of the inorganic core (120) with the radioisotope (140) interface layer completed was lipid-coated with an organic or inorganic shell (160) (e.g., polyethylene glycol). The inorganic core (120) with the radioisotope (140) interface layer completed and polyethylene glycol such as DSPE-PEG (2000) or DSPE-PEG (2000)-DBCO and NaI were added, and a microwave process was performed at 5 A, 200 W, 200°C for 5 minutes, preferably under atmospheric pressure. It was confirmed that polyethylene glycol was coated on the surface of the inorganic core (120) with the radioisotope (140) interface layer completed, and that oxidized iodine existed between the polyethylene glycol molecules. At this time, it was found that the oxidized iodine reduced the reactivity of the nanoparticles (100), thereby reducing their movement to normal organs.

[0143] 3) Attachment of target ligand (180): A covalent bond can be formed between a functional group such as DBCO of the organic shell or inorganic shell (160) coated on the inorganic core (120) and a functionalized part of an antibody such as an azide group. A functionalized antibody such as trastuzumab, into which an azide group is introduced, was added to the inorganic core (120) coated with the organic shell or inorganic shell (160), and stirred under the conditions of room temperature, pH 7, and 1 hour. Through a click chemistry reaction, it was confirmed that a covalent bond was formed between the functional groups, and that the antibody was attached to the surface of the organic shell or inorganic shell (160).

[0144] 4) Cut off and purification: By removing impurities and removing products by centrifugation and syringe filter, a radiopharmaceutical was obtained utilizing nanoparticles (100) in which a targeting ligand (180) such as trastuzumab antibody is attached to an inorganic core (120) such as iron oxide or CuI coated with an organic shell or inorganic shell (160) having an interfacial layer of a radioisotope (140) such as Lu-177 or Ac-225.

[0145]

[0146] - One embodiment of a one-step radioisotope binding and coating method

[0147] According to one embodiment of the present invention, a one-step radioisotope binding and coating method (wherein a radioisotope (140) is stabilized at the interface of an iron oxide inorganic core (120) and an organic shell or inorganic shell (160) is coated, and then a targeting ligand (180) is attached) is described.

[0148] 1) Step of coating the organic shell or inorganic shell (160) while stabilizing the inorganic core interface and attaching the radioisotope: A solution as shown in Table 1 below was added to a solution containing an inorganic core (120) containing iron oxide and CuF2, and the reaction vessel was stirred to react, and then the solvent was removed using a rotary evaporator. After adding 4 ml of distilled water, NaI and Lu-177 or Ac-225 were reacted with the CuF2 of the inorganic core (120) using an ultrasonic cleaner for 1 minute. 30 mg of nanoparticles dispersed in 1 ml of toluene per surface area of ​​the inorganic core (120) (1 nm 2 ) After adding DSPE-PEG2000 and DSPE-PEG2000-DBCO in a ratio of 1:10 at 8 ratios, NaI was added and the microwave process was performed under the conditions of 5A, 200W, 200℃, 5 minutes, and vacuum.

[0149] 2) Antibody binding step: Trastuzumab functionalized with an azide group was added and stirred at room temperature, pH 7, for 1 hour.

[0150] 3) Purification, buffer exchange, and concentration steps: The solution was centrifuged (12,000 rpm / 30 min) using a centrifuge, the precipitate was discarded, and the supernatant was collected for the first purification. The purpose of this was to remove particles that had become clumped together and had a total size of 100 nm or larger. Next, the second purification was performed once using an IEX column connected to FPLC equipment. The purified solution was washed three times with water for injection using a purification centrifuge tube and a centrifuge, and concentrated to the final concentration based on the finished product. For example, the final concentration based on the finished product is 1 mg. Fe / ml may be.

[0151] Stock AStock BIron oxideChloroformDSPE-mPEGDSPE-mPEG-FAEthanol5 mg400 μl39.6 mg0.4 mg320 μl

[0152]

[0153] According to one embodiment of the present invention, upon completion of purification, buffer exchange and concentration, a radiopharmaceutical having an antibody (Trastuzumab) attached thereto is obtained, wherein a layer of radioisotope (Lu-177 or Ac-225) is stabilized at the interface between the inorganic core and the polymer.

[0154]

[0155] - One embodiment of a multi-step radioisotope binding and coating method

[0156] According to one embodiment of the present invention, a multi-step coating method (a radioisotope (140) is stabilized at the interface of an iron oxide inorganic core (120) and an organic shell or inorganic shell (160) is coated, and then a targeting ligand (180) is attached) is described.

[0157] 1) Step of stabilizing the inorganic core interface and attaching a radioisotope: A solution such as the above [Table 1] was added to a solution containing an inorganic core (120) containing iron oxide and CuF2, and the reaction vessel was stirred to react, and then the solvent was removed using a rotary evaporator. After adding 4 ml of distilled water, an ultrasonic cleaner was used for 1 minute to react NaI and Lu-177 or Ac-225 with the CuF2 of the inorganic core (120). The microwave process was performed under the conditions of 5 A, 200 W, 200 ° C., 5 minutes, and vacuum. After separating the solution using a centrifuge (12,000 rpm / 30 min), the first purification was performed by discarding the precipitate and taking the supernatant. The purpose of this is to remove particles that have become clumped together and have a total size of more than 100 nm.

[0158] 2) Step of coating organic or inorganic shell: 30 mg of nanoparticles dispersed in 1 ml of toluene per 1 ml of inorganic core (120) surface area (1 nm 2) DSPE-PEG2000 and DSPE-PEG2000-DBCO were added in a 1:10 ratio at 8 ratios, NaI was added, and a microwave-assisted process was performed at 5 A, 200 W, 200°C, 5 minutes, and atmospheric pressure.

[0159] 3) Antibody binding step: Trastuzumab functionalized with an azide group was added and stirred at room temperature, pH 7, for 1 hour.

[0160] 4) Purification, buffer exchange, and concentration steps: The first purification was performed by centrifuging the solution (12,000 rpm / 30 min) using a centrifuge, discarding the precipitate, and collecting the supernatant. The purpose of this was to remove particles that had become clumped together and had a total size of 100 nm or larger. Next, the second purification was performed once using an IEX column connected to FPLC equipment. The purified solution was washed three times with water for injection using a purification centrifuge tube and a centrifuge, and concentrated to the final concentration based on the finished product. For example, the final concentration based on the finished product is 1 mg. Fe / ml may be.

[0161] According to one embodiment of the present invention, upon completion of purification, buffer exchange and concentration, a radiopharmaceutical having an antibody (Trastuzumab) attached thereto is obtained, wherein a layer of radioisotope (Lu-177 or Ac-225) is stabilized at the interface between the inorganic core and the polymer.

[0162]

[0163] - Another embodiment of a multi-step coating method (1)

[0164] According to one embodiment of the present invention, 30 mg of inorganic core dispersed in 1 ml of toluene per inorganic core surface area (1 nm) 2) DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]) was added in a ratio of 8 and sonicated for 1 hour. After that, toluene was removed using an evaporator, 3 ml of ethanol was added, and sonicated for 1 hour. After removing ethanol using an evaporator, it was dispersed in 1 ml of water. The residual reactant was separated using centrifugation (50,000 rpm, 1 h), and a lipid-PEG-coated iron oxide inorganic core (120) was obtained.

[0165]

[0166] Next, a method of binding Herceptin® (Roche) to a coated inorganic core (120) according to one embodiment of the present invention is described.

[0167] According to one embodiment of the present invention, 30 mg of nanoparticles dispersed in 1 ml of toluene have an inorganic core (120) surface area (1 nm). 2) DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]) and DSPE-PEG2000-amine (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]) were added at a ratio of 1:10 and sonicated for 1 hour. Toluene was removed using an evaporator, 3 ml of ethanol was added, and sonicated for 1 hour. After removing ethanol using an evaporator, it was dispersed in 1 ml of water. The remaining reactant was separated using centrifugation (50,000 rpm, 1 h), and an inorganic core (120) with an amine group introduced was obtained. After adding Sulfo-SMCC (Thermofisher Scientific) to the nanoparticles with an amine group, stirring was performed at room temperature for 1 hour, and Herceptin® (Roche) with a thiol group was added and stirred for 4 o The reaction was carried out for 12 hours in C.

[0168] Afterwards, Hiload was used to purify the antibody-bound inorganic core (120). TM The antibody-inorganic core (120) complex was obtained using FPLC (Fast Protein Liquid Chromatography) equipped with a Superdex200pg 26 / 60 (CV 318mL) column. The obtained antibody-inorganic core (120) complex was concentrated to the target concentration using a stirred cell and a 100K MWCO Amicon Centrifugal Filter, and then filtered using a filter and 4 o Stored in C.

[0169]

[0170] - Another embodiment of a multi-step coating method (2)

[0171] A multi-step coating according to one embodiment of the present invention (a method of combining Lu-177 or Ac-225 to an inorganic core (120) including iron oxide and CuF2 and stabilizing the surface of the inorganic core (120) is described.

[0172] A solution as shown in Table 2 below is added to a solution containing an inorganic core (120) including iron oxide and CuF2, and the reaction vessel is stirred to react, after which the solvent is removed using a rotary evaporator. After adding 4 ml of distilled water, NaI and Lu-177 or Ac-225 are added using an ultrasonic cleaner for 1 minute, and then reacted with the CuF2 of the inorganic core (120). After performing a microwave process under vacuum conditions of 5 A, 200 W, and 200 °C for 5 minutes, the solution is separated using a centrifuge (12,000 rpm / 30 min), and the first purification is performed by discarding the precipitate and taking the supernatant. The purpose of this is to remove particles that have become clumped together and have a total size of more than 100 nm.

[0173] Stock AStock BIron oxideChloroformDSPE-mPEGDSPE-mPEG-FAEthanol5 mg400 μl38 mg2 mg320 μl

[0174]

[0175] Next, a solution containing DIW, PEG, and a linker was added to the reaction vessel, and a microwave-assisted process was performed under atmospheric pressure, 5 A, 200 W, 200 ° C, 5 min. After that, a solution containing an antibody was added, and the reaction was carried out by stirring at room temperature for 16 hours. The solution after the reaction was completed was centrifuged using a purification centrifuge tube and a centrifuge (4000 rpm / 20 min), washed three times with distilled water, and then purified once through ion exchange chromatography connected to an FPLC equipment for secondary purification. The purified solution was washed three times with water for injection using a purification centrifuge tube and a centrifuge, and concentrated to the final concentration based on the finished product. Accordingly, when the buffer exchange and concentration were completed, radioactive core-shell nanoparticles in which a radioisotope (Lu-177 or Ac-225) layer was introduced to the inorganic core (120) interface could be obtained.

[0176]

[0177] - A method of coating an inorganic core (120) with CM (Carboxymethyl)-dextran and binding a target ligand (180) to the coated inorganic core (120) in either a single-step or multi-step manner.

[0178] According to one embodiment of the present invention, a method for coating an inorganic core (120) including a radioactive isotope layer at an interface with CM (Carboxymethyl)-dextran is described.

[0179] An inorganic core (120) containing iron oxide was dispersed in toluene using an ultrasonic bath, and a tetramethylammonium hydroxide solution (25 wt% in methanol) was mixed with the dispersed nanoparticles. The mixture was reacted in an ultrasonic bath for 1 hour, and the particles were collected using a magnet and the filtrate was discarded. The remaining particles were dispersed in distilled water. Afterwards, CM (Carboxymethyl)-dextran was dissolved in distilled water, and the pretreated inorganic core was added and heated at 70 °C for 8 hours. After the reaction was completed, the solution was transferred to a centrifuge tube and centrifuged at 3000 rpm for 10 minutes to separate the aggregated particles and collect the supernatant. An excess of ethanol was added to the separated supernatant to cause precipitation, which was then separated using a magnet. This process was repeated several times to obtain coated magnetic particles.

[0180]

[0181] Hereinafter, a method for binding a target ligand (180) to an inorganic core (120) coated with CM (Carboxymethyl)-dextran in a single step manner is described.

[0182] FIG. 8A is a diagram illustrating a single-step reaction for binding a targeting ligand to a CM (Carboxymethyl)-dextran-coated inorganic core according to some embodiments of the present invention.

[0183] 25 mg of EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide), 30 mg of S-NHS (N-hydroxysulfosuccinimide), and pH 6 MES buffer were added to the inorganic core (5 mg / ml) coated with CM (Carboxymethyl)-dextran dispersed in distilled water, and stirred at room temperature for 60 minutes. In addition, pH 7.4 PBS buffer was added, and unreacted EDC and sulfo-NHS were removed using a PD-10 desalting column. The antibody Herceptin® (Roche) was added to this, and the total volume was made 150 ml with pH 7.4 PBS buffer, and 4 o The reaction was carried out at C for 12 hours. After the reaction was completed, the mixture was concentrated using a 30K stirred cell, and the antibody-inorganic core complex was obtained using FPLC (Fast Protein Liquid Chromatography) to purify the nanoparticles bound to the antibody. The obtained antibody-inorganic core complex was concentrated to the target concentration using a stirred cell and a 100K MWCO Amicon Centrifugal Filter, and then filtered using a filter and 4 o Stored in C.

[0184] Below, a method for binding a target ligand (180) to an inorganic core (120) coated with CM (Carboxymethyl)-dextran in a multi-step manner is described.

[0185] FIG. 8b is a diagram illustrating a multi-step reaction for binding a target ligand to a CM (Carboxymethyl)-dextran-coated inorganic core according to some embodiments of the present invention.

[0186] According to one embodiment of the present invention, an inorganic core (5 mg) coated with CM (Carboxymethyl)-dextran dispersed in 1 ml of distilled water Fe / ml) and distilled water (1.5 ml) were added and stirred for 30 minutes. 15 mg of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 20 mg of N-hydroxysulfosuccinimide (S-NHS) were added and reacted at room temperature for 12 hours. Additionally, pH 7.4 PBS buffer was added and unreacted EDC and sulfo-NHS were removed using a PD-10 desalting column to obtain an inorganic core with an amine group introduced.

[0187] Afterwards, to manufacture an antibody with a thiol group introduced, Herceptin® (Roche) was added to Traut's reagent (2-Iminothiolane, Thermofisher Scientific) and treated at room temperature for 1 hour. After adding Sulfo-SMCC (Thermofisher Scientific) to the inorganic core with an amine group introduced, the mixture was stirred at room temperature for 1 hour, and Herceptin® (Roche) with a thiol group introduced was added and stirred for 4 o The reaction was carried out for 12 hours at C. Accordingly, to purify the inorganic core bound to the antibody, Hiload TM The antibody-inorganic core complex was obtained using FPLC (Fast Protein Liquid Chromatography) equipped with a Superdex200pg 26 / 60 (CV 318mL) column.

[0188] Afterwards, the obtained antibody-inorganic core complex was concentrated to the target concentration using a Stirred Cell and a 100K MWCO Amicon Centrifugal Filter, filtered, and 4 o Stored in C.

[0189]

[0190] - One example of a step of binding a target ligand (180) to the surface of a weapon core (120) using Protein A

[0191] FIG. 9 is a diagram showing a reaction of binding a target ligand using Protein A to an inorganic core coated with CM (Carboxymethyl)-dextran according to some embodiments of the present invention.

[0192] According to one embodiment of the present invention, a step of binding a target ligand (180) to the surface of an iron oxide inorganic core (120) coated with CM (Carboxymethyl)-dextran using Protein A is described.

[0193] According to one embodiment of the present invention, an iron oxide inorganic core (120) (5 mg) coated with CM (Carboxymethyl)-dextran dispersed in 1 ml of distilled water Fe / ml) and Protein A (50 mg) and 1.5 ml of distilled water were added and stirred for 30 minutes. After adding 15 mg of EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) and 20 mg of S-NHS (N-hydroxysulfosuccinimide), 4 o The reaction was carried out for 12 hours at C. In addition, pH 7.4 PBS buffer was added, and unreacted EDC and sulfo-NHS were removed using a PD-10 desalting column to obtain an inorganic core bound to Protein A. An antibody was added to the inorganic core bound to Protein A, and 4 o The reaction was carried out at C for 12 hours. Accordingly, the inorganic core bound to the antibody was purified using FPLC (Fast Protein Liquid Chromatography) and the antibody-inorganic core complex was obtained. The obtained antibody-inorganic core complex was concentrated to the target concentration using a stirred cell and a 100K MWCO Amicon Centrifugal Filter, and then filtered using a filter and 4o Stored in C.

[0194]

[0195] - HER2 / neu positive cancer cells

[0196] According to one embodiment of the present invention, the targeting ligand (180) targeting HER2 / neu expressing cancer cells may include an antibody, an antibody fragment, a nucleic acid / peptide aptamer, or a small molecule. Specifically, the antibody may be trastuzumab or pertuzumab. Trastuzumab is a monoclonal antibody that binds to domain IV of the ErbB2 receptor and can be used for the treatment of HER2-positive breast cancer, and pertuzumab is an antibody that binds to domain II of the ErbB2 receptor and prevents heterodimerization with ErbB2 and can be used for the treatment of HER2-positive breast cancer.

[0197] In addition, since antibody fragments are smaller than whole antibodies, they have the advantages of superior tissue penetration and low production costs, and therefore, various antibody fragments targeting HER2-positive cancer cells can be used as the targeting ligand (180) of the present invention. In addition, aptamers are short DNA or RNA sequences (nucleic acid aptamers) or peptide sequences (peptide aptamers) that bind to specific target molecules with high affinity and specificity, and have the advantages of being smaller in size, lower in production costs, and lower in immunogenicity compared to antibodies. Various nucleic acid / peptide aptamers targeting HER2-positive cancer cells can be used as the targeting ligand (180) of the present invention.

[0198] According to one embodiment of the present invention, since HER2 / neu positive cancer cells sometimes overexpress other ErbB receptors, cell surface membrane proteins, folate receptors, etc. in addition to HER2, ligands targeting these molecules can also be used to treat HER2 / neu positive cancer cells. For example, antibodies, antibody fragments, nucleic acid / peptide aptamers, etc. of proteins that have a positive correlation with ErbB2 expression, such as MET, MUC (Mucin 1), CD44, EpCAM (Epithelial Cell Adhesion Molecule), CXCR4 (CXC chemokine receptor type 4), ErbB1, ErbB3, etc. can be used as targeting ligands (180).

[0199] In addition, biocompatible inorganic polymers may be used as polymers, such as polyethylene glycol, polyethyleneamine, polyethyleneimine, polyacrylic acid, polymaleic anhydride, polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl amine, polyacrylamide, polyethylene glycol, phosphoric acid-polyethylene glycol, polybutylene terephthalate, polylactic acid, polytrimethylene carbonate, polydioxanone, polypropylene oxide, polyhydroxyethyl methacrylate, starch, dextran derivatives, sulfonic acid amino acids, sulfonic acid peptides, silica, polypeptides, etc.

[0200]

[0201]

[0202] - Example of attaching antibodies to nanoparticles

[0203] FIG. 10 is a drawing for explaining an example of attaching an antibody to a nanoparticle according to one embodiment of the present invention.

[0204] (1) Preparation stage (S10)

[0205] 1) Precursor synthesis stage (S11)

[0206] As illustrated, in the precursor synthesis step (S11), metal oxides and other compounds that serve as the basic materials for antibody nanoparticles were prepared. As described above, Fe-based FeCl3 hexahydrate and sodium oleate were stirred at 110°C for 8 hours in a solvent ratio of ethanol:hexane:water = 2:3:1, thereby forming the desired precursor, iron oleate compound.

[0207] 2) Purification stage (S12)

[0208] According to one embodiment of the present invention, in the purification step (S21), the precursor iron oleate mixture was subjected to layer separation using a phase separation extraction method or a centrifuge. Through this, impurities, unreacted substances, and other residues of the precursor compound were washed three times with DI water solvent through the phase separation extraction method, and additionally, 130 o The heating process was carried out for 15 hours under C conditions.

[0209] 3) Spectroscopic stage (S13)

[0210] According to one embodiment of the present invention, in the spectroscopy step (S13), analysis such as FT-IR or NMR was performed to confirm the chemical bonding state of the precursor compound. In addition, UV-Vis spectroscopy was used to confirm the optical properties of the precursor compound.

[0211] 4) Residual solvent removal and blending step (S14)

[0212] According to one embodiment of the present invention, in the residual solvent removal and blending step (S14), the oleic acid iron solution obtained in the purification step (S12) was degassed for 3 hours to remove unnecessary residual solvent and low molecular weight impurities within the mixture. In addition, a solvent of octadecene and oleyl alcohol in a ratio of 5:3 was added to the precursor mixture from which the residual solvent had been removed, and a blending process was performed. In the blending process, ultrasonication, heating, degassing, or mechanical shaking was utilized to increase stability so that layer separation of the solution did not occur.

[0213] 5) Spectroscopy and storage stage (S15)

[0214] According to one embodiment of the present invention, FT-IR or NMR analysis was performed to confirm the chemical bonding state of the precursor mixture in the spectroscopy and storage step (S15). Specifically, specific bonds of Fe-based oxides (e.g., Fe-O) and functional groups of organic compounds (e.g., C=O, CH, etc.) were confirmed, and the analysis results confirmed that the precursor met the desired chemical properties. In addition, appropriate conditions were set for storing the precursor mixture after the analysis was completed, and it was confirmed that the chemical stability of the particles was maintained by storing it under conditions of 4°C to 30°C and a relative humidity of 70% or less. In this case, the precursor mixture was stored in a sealed container.

[0215]

[0216] (2) Nanoparticle generation stage (S20)

[0217] 1) Nanoparticle manufacturing and radioisotope stabilization step (S21)

[0218] As illustrated, the nanoparticle manufacturing and radioisotope stabilization step (S21) is a step of manufacturing nanoparticles (100) with CuF2 introduced at the interface, and forming a stabilizing layer while introducing a radioisotope (140) to the nanoparticles. In this step, nanoparticles (100) were formed using metal oxides and coating materials, and a layer of Lu-177 or Ac-225, which is a radioisotope (140), could be formed at the interface. Specifically, the reaction was initiated by mixing materials such as Fe-based metal oxides (including CuF2 doping) and DSPE-PEG(2000), DSPE-PEG(2000)-DBCO, and radioactive isotopes Lutetium-177 (260 mCi or Actium-225 (250 mCi). The mixture was subjected to a microwave-assisted process using microwave equipment (CEM Discover 2.0) at 5 A, 200 W, and 200°C for 5 minutes to form uniform nanoparticles (100), preferably under vacuum conditions. Subsequently, an ultrasonic dispersion process was performed to increase the size and uniformity of the nanoparticles and prevent aggregation between particles. Afterwards, radioactive Lu-177 or Actium-225 was able to form a stabilized layer at the interface of the nanocore.

[0219] 2) Centrifugation step (S22)

[0220] As illustrated, the centrifugation step (S22) is a process for removing impurities and separating only the desired nanoparticles in the step of generating nanoparticles in which radioactive isotopes are stabilized at the interface. The present invention separates nanoparticles by spinning at 10,000 rpm or more using a high-speed centrifuge, and it was confirmed that the separation time was maintained for about 1 hour. In order to remove residual impurities during the purification process, the material was washed using an appropriate solvent such as ethanol (Ethyl alcohol) and hexane. The washing process was performed repeatedly, and the supernatant was removed and only the sediment was passed to the filtration and evaporation step (S23).

[0221] 3) Filtration and evaporation stage (S23)

[0222] As shown, in the filtration and evaporation step (S23), nanoparticles were filtered using an Amicon filter (pore size 0.2 μm or less) or other ultrafine filter. A small amount of hexane was added to the sediment to disperse it, followed by filtration. The solvent was removed from the filtered nanoparticle solution using low-pressure conditions (e.g., a reduced-pressure evaporator). In this case, the solvent was completely removed using a rotary evaporator or freeze dryer.

[0223] 4) Concentration adjustment and QC_DLS analysis step (S24)

[0224] As shown, in the concentration adjustment and QC_DLS analysis step (S24), the final concentration was adjusted by adding an appropriate solvent such as toluene to the concentrated nanoparticle solution obtained in the filtration and evaporation step (S23). Specifically, the concentration of nanoparticles (100) was 1 mg. Fe / mL was adjusted. In addition, the particle size distribution was calculated by measuring the particle movement speed by transmitting a laser through the nanoparticles (100) through DLS analysis. At this time, it was confirmed that the average particle size was within the range of 20 to 50 nm. Since the PDI value was maintained below 0.3, it could be evaluated that the particles were uniform.

[0225] 5) Ion chromatography and storage step (S25)

[0226] As illustrated, the ion chromatography and storage step (S25) was performed by passing the nanoparticle solution through a stationary phase, such as an ion exchange column. Within the column, ionic substances interacted with the stationary phase to separate, and the nanoparticles were recovered by passing through the column along with the mobile phase. The nanoparticle solution subjected to ion chromatography was highly purified, with impurities removed. The purified nanoparticles were stored under refrigerated conditions at 4°C to 8°C.

[0227]

[0228] (3) Linker attachment step (S30)

[0229] 1) RI / Ab linker conjugation and organic polymer shell or inorganic polymer shell coating step (S31)

[0230] As shown, the RI / Ab linker conjugation and organic polymer shell or inorganic polymer shell coating step (S31) is a step of conjugating a linker to connect nanoparticles (RI-doped nanoparticles) and antibodies (Antibody, Ab) and a step of coating an organic or inorganic polymer shell. In the RI / Ab linker conjugation and organic polymer shell or inorganic polymer shell coating step (S31), PEG was used as a material for the organic polymer shell or inorganic polymer shell. DBCO-PEG (dibenzocyclooctane-PEG) was used as a linker molecule (182). This is a linker utilizing Cu-free Click Chemistry and was used to connect RI-doped nanoparticles and antibodies. A mixture of PEG, DBCO-PEG, and nanoparticles was subjected to a microwave reaction at atmospheric pressure for 5 A, 200 W, and 200°C for 5 minutes using microwave equipment (CEM Discover 2.0). Additionally, a maleimide-based linker was used as a linker molecule (182). This formed a bond by reacting with the thiol group (-SH) of the antibody molecule (181) of the present invention.

[0231]

[0232] (4) Antibody attachment step (S40)

[0233] 1) Antibody functionalization step (S41)

[0234] As illustrated, the antibody functionalization step (S41) is a step for replacing the solvent of the antibody molecule (181) to be used to suit the reaction conditions and for removing impurities or residual salts that may affect the reaction before manufacturing the nanoparticle-antibody binding complex (200). A desalting column was functionalized using the size-exclusion chromatography (SEC) principle, and an ultrafiltration filter (Amicon Ultra Filter) with a pore size of 10 to 30 kDa was used to inject an antibody solution such as trastuzumab into the column, and the process of eluting through a mobile phase (PBS or DIW) was performed.

[0235] 2) Antibody linker conjugation step (S42)

[0236] As described above, the functionalized antibody was chemically conjugated to a linker to prepare an antibody-linker complex. In the antibody-linker conjugation step (S42), the pH can be maintained at 6.5 to 7.5 depending on the type of linker molecule (182), and an antibody solution, such as trastuzumab, was mixed with the linker solution and stirred (50 to 100 rpm) to conjugate the antibody molecule (181) and the linker molecule (182). Thereafter, chromatography or filtration was performed to remove unreacted linker molecules and impurities from the antibody solution after linker conjugation was completed.

[0237] 3) SEC purification and storage stage (S43)

[0238] As illustrated, the SEC purification and storage step (S43) utilizes Size-Exclusion Chromatography (SEC) technology to remove residual impurities (unreacted linker, antibody, small molecules, etc.) from the antibody-linker complex after the antibody-linker conjugation step (S42). The antibody-linker complex solution was injected into the column, and the mobile phase was allowed to flow to separate the complexes based on differences in molecular size. Accordingly, the large antibody-linker complex eluted first, and since small molecules remained inside the column longer, only the eluted fractions containing the complex were selectively collected. The purified antibody-linker complex was stored dispersed in PBS or DIW solution in a refrigerated environment at 4°C to 8°C.

[0239] 4) Antibody nanoparticle conjugation step (S44)

[0240] As illustrated, the antibody-nanoparticle conjugation step (S44) is a step in which the antibody-linker complex purified in the SEC purification and storage step (S43) or the primary concentration, purification, and storage step (S35) is combined with nanoparticles to generate the final antibody-nanoparticle complex. Specifically, purified nanoparticles, such as radioactive Lutetium-177-doped nanoparticles, were dispersed in an appropriate solvent (PBS or DIW) to prepare a uniform state, and functional groups on the nanoparticle surface were activated (e.g., DBCO, Maleimide, etc.) to promote binding to the antibody-linker. Thereafter, the antibody-linker complex solution was added to the nanoparticle solution. In this case, the reaction conditions were carried out at 20-25°C (room temperature), and the pH was maintained at 6.5-7.5, stirring was performed at 150 RPM, and the process was performed for approximately 1 hour.

[0241] 5) Concentration and SEC purification step (S45)

[0242] As illustrated, the concentration and SEC purification step (S45) is a step to remove unreacted substances and impurities from the antibody-nanoparticle complex generated after the antibody-nanoparticle conjugation step (S44), and to adjust the complex to an appropriate concentration. The solvent was removed and concentrated using an ultrafiltration filter with a pore size of 10–30 kDa, and the complex was filtered through low-speed centrifugation (3,000–4,000 rpm) to perform the concentration. In addition, the concentration of the nanoparticle-antibody conjugated complex (200) was optimized (e.g., 1–10 mg / mL) by adding PBS (phosphate-buffered saline) or DIW (deionized water). To remove unreacted substances and impurities from the concentrated complex and produce a high-purity nanoparticle-antibody complex (200), a chromatography system (FPLC or HPLC) and an SEC column such as Superdex 200 or Sephadex G-25 were used to separate the complex and small molecules based on their size differences. Accordingly, the nanoparticle-antibody complex (200) was eluted first due to its large molecular size, and unreacted substances were eluted later.

[0243] 6) Concentration and purification stage (S46)

[0244] As illustrated, the concentration and purification step (S46) is performed after the concentration and SEC purification step (S45), and is a step to further concentrate the antibody-nanoparticle complex and ultimately remove residual impurities (unreacted substances, solvents, small particles, etc.). Fine particles and insoluble impurities were removed using a membrane filter (pore size of 0.22 μm or less), and high-purity purification was performed through additional Size-Exclusion Chromatography (SEC) or Ion Exchange Chromatography.

[0245] 7) QC and storage stage (S47)

[0246] As shown, the QC (Quality Control) and storage step (S47) is the final step to inspect and store the quality of the antibody-nanoparticle complex after the concentration and purification step (S46). The average size (20–50 nm) and size distribution (PDI: Polydispersity Index) of the nanoparticles were measured to ensure homogeneity with a PDI of 0.1–0.2 or less. HPLC / FPLC or SDS-PAGE was used to confirm whether unreacted substances (antibody, nanoparticles, linker) and impurities within the complex were removed. In addition, the chemical bonding state between the antibody and nanoparticles was verified through UV-Vis and FT-IR analyses, and whether the target binding ability of the antibody was maintained was confirmed through ELISA or target binding test. Finally, the antibody-nanoparticle complex after completion of purification and QC was stored.

[0247]

[0248] - Schematic diagram of antibody functionalization step (1)

[0249] Figure 11 is a schematic diagram showing an antibody functionalization step according to one embodiment of the present invention.

[0250] As illustrated in FIG. 11, the antibody attachment step (S40) of the present invention may include an antibody functionalization step (S41). Specifically, if the working environment (buffer, salt concentration, etc.) of the antibody molecule (181) is inappropriate, the binding reaction with the linker molecule (182) may be inefficient or unstable. Therefore, the antibody functionalization step (S41) replaces the antibody molecule (181) with a new buffer suitable for the reaction, thereby optimizing the reaction environment, such as pH or ionic concentration, so that the linker molecule (182) and the antibody molecule (181) effectively bind. In addition, the antibody molecule (181) may contain other impurities, such as salts, preservatives, or stabilizers, during the manufacturing and storage processes. Since such impurities may interfere with the chemical reaction or reduce the efficiency of the linker-antibody binding, the efficiency of the binding reaction with the linker molecule (182) is increased by removing salts and other low-molecular-weight substances from the antibody molecule (181) in the antibody functionalization step (S41).

[0251] In addition, an antibody molecule (181) containing an amine (-NH2) functional group was combined with a linker molecule (182) containing maleimide and azide functional groups. Here, the linker molecule (182) serves to connect (link) between the antibody molecule (181) and the nanoparticle (100), and acts as a chemical bridge to stably combine the antibody molecule (181) and the nanoparticle (100). Here, it was confirmed that the maleimide functional group of the linker molecule (182) formed an amide bond by bonding with the amine (-NH2) group of the antibody molecule (181), thereby fixing the antibody molecule (181) and the linker molecule (182). Accordingly, an antibody (183) chemically bonded with the linker molecule was generated, and the antibody (183) chemically bonded with the linker molecule could have a form capable of reacting with the nanoparticle (100).

[0252]

[0253] - Schematic diagram of antibody functionalization step (2)

[0254] Figure 13 is a schematic diagram showing an antibody functionalization step of attaching a thiol functional group to an antibody according to one embodiment of the present invention.

[0255] As illustrated, a linker molecule (182) having a 2-iodoacetamide-like structure is bound to an antibody molecule (181) to attach a thiol functional group to the antibody molecule (181). Specifically, the linker molecule (182) contains a thioredoxin structure (S and NH functional groups), and thus binds to the antibody molecule (181) and attaches a highly reactive thiol group. In this case, the antibody molecule (181) and the linker molecule (182) can bind through a site-specific reaction, and the functional groups (S and NH) of the linker react with a specific site of the antibody to form a covalent bond. As a result, a thiol (-SH) functional group can be attached to the terminal when the linker is attached to the antibody. Finally, an antibody (183) chemically bound to the linker molecule having a thiol (-SH) functional group attached to the surface of the antibody molecule is generated.

[0256]

[0257] - Covalent bond

[0258] According to one embodiment of the present invention, the process of forming a covalent bond between a linker functional group and a specific portion of an antibody can be performed based on the structural characteristics of the antibody molecule (181) and the chemical characteristics of the linker molecule (182). Specifically, the antibody can include a reactive functional group such as an amine group (-NH2) of a lysine residue, a carboxyl group (-COOH) of a glutamic acid or aspartic acid residue, or a sulfhydryl group (-SH) of a cysteine ​​residue. The linker molecule (182) has a chemical structure designed to selectively react with such specific functional groups, and the chemical reaction between the linker molecule (182) and the antibody molecule (181) can be performed with high efficiency and selectivity.

[0259] According to one embodiment of the present invention, the binding process between the antibody molecule (181) and the linker molecule (182) may proceed through the following mechanism. First, when the antibody molecule (181) and the linker molecule (182) are mixed under appropriate pH and temperature conditions, the maleimide functional group included in the linker molecule (182) can rapidly react with the cysteine ​​sulfhydryl group (-SH) of the antibody molecule (181) to form a thioether bond. This reaction proceeds as an addition-dehydrogenation reaction and can have high selectivity. Meanwhile, when the linker molecule (182) includes an N-hydroxysuccinimide (NHS) ester, it can react with the amine group of the lysine residue to form an amide bond (-CO-NH-). This proceeds as a nucleophilic substitution reaction, and the generated amide bond can be stable even under physiological conditions.

[0260] According to one embodiment of the present invention, the linker molecule (182) is designed to maintain the structural stability and biological activity of the antibody molecule (181) during the binding process, so that the antibody molecule (181) can perform its original biological function even after modification. In addition, through covalent bonding with the linker molecule (182), the antibody can have reactivity, such as a thiol group (-SH) required for subsequent reactions.

[0261]

[0262] - Non-covalent bond

[0263] According to the present invention, the process of forming a non-covalent bond between a linker functional group and a specific region of an antibody can be achieved through a physical interaction instead of a chemical covalent bond. In this case, the antibody molecule (181) is composed of a protein and thus can provide various interaction sites, such as a hydrophobic region, a charged region, and a hydrogen bonding site. The linker molecule (182) has a chemical structure designed to form a non-covalent bond with the antibody molecule (181), and the hydrophobic domain, charged functional group, or hydrogen bond contributing functional group of the linker molecule (182) can induce binding to a specific region of the antibody molecule (181).

[0264] According to the present invention, non-covalent bonds can be formed through three mechanisms. First, hydrophobic interactions may involve a process in which hydrophobic amino acids on the surface of an antibody molecule (181) and hydrophobic domains of a linker come close to each other, thereby excluding water molecules and forming a stable bond. Second, electrostatic attraction is formed by the difference in charge distribution between the linker molecule (182) and the antibody molecule (181). If the linker molecule (182) is positively charged, it can bind to the negatively charged portion of the antibody molecule (181), or conversely, if the linker molecule (182) is negatively charged, it can bind to the positively charged portion of the antibody molecule (181). Third, hydrogen bonds may form a strong non-covalent bond by sharing hydrogen atoms between the linker molecule (182) and the antibody molecule (181). In other words, non-covalent bonds are more reversible than covalent bonds, and bonds can be formed or dissociated depending on the surrounding conditions (pH, ionic concentration, temperature, etc.). However, by optimizing conditions, stability can be increased, and the antibody and linker can form a close complex through binding with specificity.

[0265]

[0266] - Production of antibodies chemically bound to linker molecules using linker molecules containing NHS (N-hydroxysuccinimide) esters

[0267] FIG. 15 is a diagram illustrating a step of generating an antibody chemically bound to a linker molecule using a linker molecule including an NHS ester according to one embodiment of the present invention.

[0268] As illustrated, the antibody molecule (181) is a biomolecule composed of proteins and has a biological recognition function capable of binding to a specific target, and in this process, the amine group (-NH2) of the lysine residue can be used for the reaction. The NHS ester linker (182), another key component of the reaction, can include an NHS ester functional group that can selectively react with the amine group. The linker molecule (182) can perform a multifunctional role by maintaining a terminal functional group (e.g., azide, etc.) for further reaction even after binding to the antibody molecule (181).

[0269] According to the present invention, the reaction between an antibody molecule (181) and a linker molecule (182) can proceed as a nucleophilic substitution reaction. First, an NHS ester linker and an antibody are mixed under appropriate conditions, and a mildly acidic to neutral environment of pH 7.0 to 8.5 can promote the reaction between the NHS ester and the amine group of the antibody. During the reaction, the amine group of the antibody attacks the activated carbonyl carbon of the NHS ester, causing the hydroxysuccinimide (NHS) moiety to detach, and an amide bond (-CO-NH-) to form between the antibody and the linker. The reaction product is an antibody-linker complex (183) in which the antibody and the linker are stably covalently bonded, and in this complex, the antibody maintains biological activity and can bind to a specific target.

[0270]

[0271] - Antibody nanoparticle conjugation (S44)

[0272] (1) One example

[0273] FIG. 12 is a diagram showing a step of producing a nanoparticle-antibody (trastuzumab) binding complex according to one embodiment of the present invention.

[0274] As shown in Fig. 12, IONP(177-Lu)-DBCO is a starting material, and a radioactive isotope (140) lutetium-177( is added to an Iron Oxide Nanoparticle, which is an example of a nanoparticle (100).177 Lu) or 225 Ac was included. Here, DBCO (Dibenzylcyclooctyne) is a chemical functional group linked to IONP, and it contains a cyclooctyne structure with high reactivity. The antibody (183) chemically bonded to the linker molecule contains a specific azide (N3) functional group, and the azide (N3) functional group is designed for binding to DBCO. The bond is formed through the azide functional group on the surface of the antibody molecule (181), which enables binding to nanoparticles while maintaining the biological activity of the antibody.

[0275] According to the present invention, Click Chemistry can be used in the antibody nanoparticle conjugation step (S44). Click Chemistry is a highly efficient and selective reaction mechanism. DBCO (DSPE-PEG2000-DBCO (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carbonyl-amino(polyethylene glycol)-2000-N'-(succinylazadibenzocyclooctyne)] (ammonium salt)) and azide (N-Succinimidyl 15-Azido-4,7,10,13-tetraoxapentadecanoate) were stirred for 2 hours in a pH 7.4 PBS:DMSO=9:1 solvent condition to form a 1,2,3-triazole bond, thereby binding the antibody and the nanoparticle. Finally, a nanoparticle-antibody binding complex (200) in which the nanoparticle and the antibody are chemically bound is formed. Confirmed.

[0276] (2) Another example

[0277] FIG. 14 is a drawing showing a step of generating a nanoparticle-antibody binding complex using an antibody containing a thiol group (-SH) according to one embodiment of the present invention.

[0278] As shown, a nanoparticle-antibody binding complex (200) can be formed through a selective reaction between maleimide and thiol, and the main starting materials used here were IONP(177-Lu)-Maleimide and Thiol-Antibody.

[0279] According to one embodiment of the present invention, IONP(177-Lu)-Maleimide is an iron oxide nanoparticle (IONP, 100) containing radioactive isotope ruthenium-177 (177-Lu), which can be utilized for diagnostic and therapeutic purposes. A maleimide functional group is linked to the surface of the nanoparticle (100), and the maleimide functional group may have a chemical property that can selectively react with a thiol group (-SH) of an antibody (183) chemically bound to a linker molecule. To this end, nanoparticles introduced with DSPE-PEG2000-Maleimide (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carbonyl-amino(polyethylene glycol)-2000-N'-(3-maleimidopropionyl)] (ammonium salt)) and antibodies introduced with a thiol group (-SH) obtained through the reaction of 2-Iminothiolane and antibodies were combined by stirring at room temperature for 2 hours in a pH 7.4 PBS solvent condition.

[0280] An antibody (183) chemically bound to a linker molecule is a biological recognition molecule capable of binding to a specific target. In this process, the antibody may be provided in the form of a Thiol-Antibody with a thiol group introduced. Here, the thiol group is provided from a cysteine ​​residue of the antibody and acts as a site for forming a covalent bond by chemically reacting with maleimide.

[0281] According to one embodiment of the present invention, the binding process of FIG. 14 may proceed primarily by a Michael Addition reaction. The maleimide functional group reacts with the thiol group of the antibody (183) chemically bound to the linker molecule to form a covalent bond, and this reaction can form a thioether bond (SC bond) as the thiol group is added to the double bond of the maleimide. The reaction proceeds efficiently in an environment of pH 6.5 to 7.5, and is usually completed within a few hours at room temperature or slightly higher. The final product, the IONP-Antibody complex (200), is a form in which the nanoparticle and the antibody are chemically bound, and it was confirmed that it can be utilized for diagnosis and treatment by combining the characteristics of the nanoparticle while maintaining the target recognition ability of the antibody.

[0282] (3) Another example

[0283] FIG. 16 is a drawing showing a step of generating a nanoparticle-antibody binding complex using an antibody containing tetrazine according to one embodiment of the present invention.

[0284] As shown, IONP(177-Lu)-TCO is a form of iron oxide nanoparticles containing radioactive isotope ruthenium-177 (177-Lu), which can be used in medical diagnosis and radiotherapy. A TCO (trans-Cyclooctene) functional group is bonded to the surface of the nanoparticle (100), and the TCO (trans-Cyclooctene) functional group has chemical properties that can selectively react with tetrazine. Another key element of the reaction, Tetrazine-Antibody, is a form in which a tetrazine functional group is attached to an antibody with target recognition function, and the tetrazine can have a ring structure that can react with TCO to form a stable covalent bond.

[0285] According to the present invention, the reaction process starts with a Tetrazine-TCO reaction, and when IONP(177-Lu)-TCO and Tetrazine-Antibody(183) are mixed, the tetrazine ring structure reacts with the ring structure of TCO to form a bond quickly and with high selectivity. This reaction proceeds without a metal catalyst, and non-specific binding with other molecules does not occur even in a biological environment. As a result of the reaction, a covalent bond is formed between the tetrazine and TCO, and a complex (200) in which an antibody and a nanoparticle are linked can be generated. It was confirmed that the finally generated IONP-Antibody complex (200) is a form in which the radioactive ruthenium-177 of the nanoparticle and the target recognition function of the antibody are combined.

[0286]

[0287] - Results of nanoparticle bonding material size analysis (DLS)

[0288] FIG. 17 is a diagram showing the results of size analysis (DLS) of an anti-Her2 nanoparticle conjugate according to one embodiment of the present invention.

[0289] Figure 17a shows the results of analyzing the particle size of the anti-Her2 antibody and nanoparticle (IONP) conjugate complex using Dynamic Light Scattering (DLS). As shown, the analysis results show that the Z-Average value is 23.48 nm, which represents the average size of the complex particles, and this falls within the standard range of 20 ± 5 nm, thus satisfying the designed size condition. In addition, the Polydispersity Index (PDI) value was very low at 0.118, confirming that the particle size distribution of the sample was uniform.

[0290] As shown, the particle size distribution exhibited a single peak at approximately 19.01 nm, indicating a single distribution and highly uniform particle size. The intercept value (0.941) and data quality assessment result ("Good") indicate that the measured data are reliable, confirming that the Anti-Her2-nanoparticle complex meets the design requirements in terms of size.

[0291] Figure 17b shows the results of analyzing the particle size of the anti-Her2 antibody-nanoparticle conjugate complex measured by the intensity method. The intensity method is based on the principle that light scattering intensity varies depending on the size of the particle, and can be used to evaluate particle size and distribution. According to the analysis results, the Z-Average value was measured to be 23.48 nm, which corresponded to the average size of the complex particles within the standard range (20 ± 5 nm). The main peak (26.49 nm) represented the most dominant particle size in the sample, confirming that the size distribution was centered around this value. In addition, the PDI (Polydispersity Index) value was 0.118, confirming that the particle size distribution was uniform.

[0292] Specifically, a lower PDI value indicates a narrower size distribution and a more stable synthesis process. The Intercept value, which evaluates data quality, was 0.941, indicating high reliability, and the overall result quality was evaluated as "Good," indicating that the measured data were accurate and reliable. In addition, the graph showing the size distribution showed a single peak located at approximately 26.49 nm and a narrow distribution width, confirming that the Anti-Her2-nanoparticle complex met the design requirements in terms of size and uniformity.

[0293]

[0294] - Analysis of Anti-Her2-nanoparticle conjugate complex using cryo-TEM (cryo-electron microscopy)

[0295] Figure 18 is a diagram showing the results of an analysis of an Anti-Her2-nanoparticle conjugate complex using Cryo-TEM (cryo-electron microscopy) according to one embodiment of the present invention. Figure 19 is a diagram showing the results of an Anti-Her2 antibody binding analysis using ELISA according to one embodiment of the present invention.

[0296] As shown, the formation status of the anti-Her2 antibody-nanoparticle (IONP) conjugate complex was visually and quantitatively analyzed using cryo-TEM (cryo-electron microscopy) and ELISA. Specifically, cryo-TEM images were used to confirm the microstructure of the complex, and the amount of anti-Her2 antibody (Trastuzumab) bound to the nanoparticles was quantitatively measured by ELISA.

[0297] According to one embodiment of the present invention, the size and structural characteristics of nanoparticles can be confirmed through cryo-TEM analysis. As shown, the size range of nanoparticles (100) is about 4.0 to 5.5 nm, the average size of nanoparticles (100) is about 4 nm, and the length of the anti-Her2 antibody (183) bound to the surface of the nanoparticles (100) is about 2.5 nm. That is, through FIG. 18, it was confirmed that the anti-Her2 antibody (183) was uniformly attached to the surface of the nanoparticles (100). In addition, it was confirmed that the nanoparticle-antibody binding complex (200) maintained a structurally stable form even after binding.

[0298] As shown in Fig. 19, the ELISA analysis confirmed the results of measuring the binding amount of trastuzumab at a concentration of 2 mg / mL of nanoparticles. The binding amount of trastuzumab in the seven experimental groups ranged from 3.62 to 4.36 mg / mL, and the average binding amount was calculated to be approximately 4.03 mg / mL. The deviation of the binding amount of trastuzumab results from Examples 1 to 7 was small and the reproducibility between the experimental groups was high, confirming that the binding process between the nanoparticles (100) and the antibody (183) was performed effectively.

[0299]

[0300] - Evaluation of plasma stability of nanoparticle-antibody complexes in serum

[0301] Figure 20 is a drawing showing the results of evaluating the serum stability (plasma stability) of a nanoparticle-antibody binding complex according to one embodiment of the present invention in various biological species (human, mouse, rat, beagle).

[0302] As shown, the results of evaluating the stability of the nanoparticle-antibody binding complex (200) in serum for various biological species (Human, Mouse, Rat, Beagle) were confirmed. The experiment of Fig. 20 was conducted to confirm whether the nanoparticle-antibody binding complex (200) is maintained without being decomposed over time in serum, and the x-axis (horizontal axis) represents the time (0 to 24 hours) that the nanoparticle-antibody binding complex (200) was exposed to serum, and the y-axis (vertical axis) represents the rate at which the nanoparticle-antibody binding complex (200) maintains its initial state as a percentage.

[0303] Looking at the results, it was confirmed that the proportion of the nanoparticle-antibody binding complex (200) that maintained the initial state in all species at the 0 hour point was approximately 100%, and that the stability was maintained in the range of 90-100% in all species even at the 5 hour point over time. Accordingly, it was confirmed that the nanoparticle-antibody binding complex (200) was not easily decomposed in the biological environment. In addition, from 10 hours to 24 hours, all nanoparticle-antibody binding complexes (200) in Human, Beagle, Mouse, and Rat still maintained a high proportion of maintaining the initial state of more than 90%, and it was confirmed that the complexes of Human and Beagle were particularly stable, maintaining values ​​close to 100%. Accordingly, it was confirmed that the nanoparticle-antibody binding complex (200) maintained a high residual proportion in the serum environment regardless of the species.

[0304]

[0305] - Confirmation of cellular uptake of antibody-conjugated nanoparticles in breast cancer cells

[0306] FIG. 21 is a drawing showing an image taken using a fluorescence microscope to confirm cellular uptake of antibody-conjugated nanoparticles (Lu177-IONP-Trastuzumab-Fluor545) in breast cancer cells (BT-474 and MDA-MB-231) according to one embodiment of the present invention.

[0307] As shown, fluorescence signals under two conditions (LIF and LITF) were observed for each of the two cell lines, BT-474 (21-1) and DA-MB-231 (21-2). The LIF condition confirmed the fluorescence signal appearing from the nanoparticles (Lu177-IONP-Fluor545) to which the antibody was not bound, and the LITF condition confirmed the fluorescence signal appearing from the nanoparticles (Lu177-IONP-Trastuzumab-Fluor545) to which the antibody was bound. In addition, the location of the nanoparticles (100) could be confirmed through red fluorescence (Fluor545, 21-3), and the location of the cells could be confirmed by staining the cell nucleus through blue fluorescence (DAPI, 21-4). The image combining the red and blue fluorescence allowed us to confirm whether the nanoparticles (100) were absorbed into the cells.

[0308] As shown, in BT-474 (HER2-positive cell line, 21-1), it was confirmed that the LITF condition (21-1b) showed a stronger red fluorescence signal than the LIF condition (21-1a). This meant that the antibody-conjugated nanoparticles (100) specifically bound to HER2-positive cells and were absorbed more. In addition, it was confirmed through the merged image that the nanoparticles (100) were located around the cell nucleus and absorbed into the cell. On the other hand, in MDA-MB-231 (HER2 low-expressing cell line, 21-2), the LITF condition (21-2b) showed a stronger fluorescence signal than the LIF condition (21-2a), but the fluorescence signal intensity was lower than that of BT-474 (21-1). This was interpreted to mean that the target specificity of trastuzumab is low in HER2-negative cells, resulting in relatively less absorption of the nanoparticles (100). That is, in HER2-positive cells such as BT-474 (21-1), it was confirmed that trastuzumab-conjugated nanoparticles (100) were more efficiently absorbed into cells due to HER2 targeting specificity, and relatively low absorption was observed in MDA-MB-231 (21-2), a HER2 low-expression cell line.

[0309] These results indicated that trastuzumab could efficiently increase the intracellular delivery of nanoparticles (100) in HER2 target cells, and confirmed the potential use of nanoparticle-antibody conjugates (200) in the treatment of HER2-overexpressing cancers. In addition, DAPI + Fluor545(21-5) was merged into an image, and red fluorescence (nanoparticles) and blue fluorescence (cell nuclei) were combined to confirm whether nanoparticles were present inside cells.

[0310]

[0311] - Comparison of the degree of intracellular uptake of antibody-conjugated nanoparticles and antibody-free nanoparticles in breast cancer cell lines.

[0312] FIG. 22 is a drawing showing the results of comparing the degree of intracellular uptake of antibody-conjugated nanoparticles (Lu177-IONP-Trastuzumab-Fluor545) and antibody-free nanoparticles (Lu177-IONP-Fluor545) in BT-474 (HER2 positive cell line) and MDA-MB-231 (HER2 low-expressing cell line) according to one embodiment of the present invention.

[0313] As shown, the degree of intracellular uptake was evaluated based on the mean fluorescence intensity (MFI) on the y-axis, which allowed us to confirm the effect of trastuzumab binding on cellular uptake.

[0314] According to the present invention, BT-474 (21-1) is a HER2-positive cell line, a cell in which HER2 receptors are overexpressed, and in the case of non-antibody-bound nanoparticles (Lu177-IONP-Fluor545, 22-1), it was confirmed that the average fluorescence intensity was about 1.0 Х 108 or less, indicating a low degree of absorption. On the other hand, it was confirmed that the average fluorescence intensity of the trastuzumab-bound nanoparticles (Lu177-IONP-Trastuzumab-Fluor545, 22-2) was about 2.5 Х 108 or more, which was about 2.5 times higher than that of the non-antibody-bound nanoparticles (22-1). Accordingly, in the present invention, it was confirmed that trastuzumab specifically binds to the HER2 receptor and significantly increases the intracellular uptake of the nanoparticles (100). In addition, the difference between the two groups (21-1, 21-2) shown in Fig. 22 was statistically highly significant (p < 0.01), confirming that trastuzumab had high target specificity in HER2-expressing cells.

[0315] According to the present invention, it was confirmed that MDA-MB-231(21-2) is a HER2 low-expression cell line, and HER2 receptors are not expressed or are expressed at a very low level. The average fluorescence intensity of the non-antibody-conjugated nanoparticles (Lu177-IONP-Fluor545, 22-1) was about 1.0 х 108 or less, and the average fluorescence intensity of the antibody-conjugated nanoparticles (Lu177-IONP-Trastuzumab-Fluor545, 22-2) was about 1.5 х 108. In other words, although it showed a fluorescence intensity about 1.5 times higher than that of the non-antibody-conjugated nanoparticles, it was confirmed that the absorption efficiency was low compared to BT-474(21-1), a HER2-positive cell line.

[0316] This is because the targeting specificity of trastuzumab is limited in HER2 low-expressing cells. In conclusion, it was confirmed that trastuzumab-conjugated nanoparticles (22-2) significantly increased intracellular uptake by targeting the HER2 receptor in HER2-positive cells (BT-474, 21-1). Although the uptake efficiency was relatively low in HER2 ultra-low-expressing cells (MDA-MB-231, 21-2), the trastuzumab-conjugated nanoparticles were absorbed very little as expected. Through these experimental results, it was confirmed that the trastuzumab-conjugated nanoparticles (22-2) of the present invention can be effectively utilized in various fields from the treatment of HER2 high-expressing to ultra-low-expressing cancers.

[0317]

[0318] - Comparison of the anticancer effects of antibody-conjugated nanoparticles and antibody-free nanoparticles in breast cancer cells.

[0319] FIG. 23 is a diagram showing the results of an In vitroEfficacy Evaluation experiment comparing the anticancer effects of antibody-conjugated nanoparticles (Lu177-IONP-Trastuzumab) and antibody-free nanoparticles (Lu177-IONP) according to one embodiment of the present invention on breast cancer cells (BT-474 and MDA-MB-231).

[0320] As shown, the experiment in Figure 23 measured cell viability (Cell viability, %) according to the concentration of radioactive nanoparticles (Log(kBq / mL)) using the CCK8 assay, and confirmed the effect of trastuzumab binding on the anticancer effect.

[0321] As shown in (a) of Figure 23, BT-474 (21-1) is a HER2-positive cell line, a cell in which HER2 receptors are overexpressed. In the case of antibody-free nanoparticles (Lu177-IONP, 23-1), it was confirmed that cell viability gradually decreased as the concentration of nanoparticles increased. Here, IC 50 The concentration of silver nanoparticles (100) required to reduce cell viability by 50% could be referred to as IC. 50 A lower value could mean that the substance is more potent, as it could reduce cell viability by half even at a lower concentration. In other words, the nanoparticles conjugated with trastuzumab (Lu177-IONP-Trastuzumab, 23-2) significantly reduced cell viability by targeting the HER2 receptor, and the IC 50 The value was approximately 377.5 kBq / mL, confirming that the same anticancer effect was observed at a much lower concentration than that of antibody-free nanoparticles (23-1). This result indicated that trastuzumab specifically binds to the HER2 receptor, significantly enhancing its anticancer effect.

[0322] As shown in (b) of Figure 23, MDA-MB-231 (21-2) is a HER2 low-expressing cell line, in which HER2 receptors were not expressed or were expressed at very low levels. In the case of antibody-free nanoparticles (Lu177-IONP, 23-1), cell viability decreased with increasing concentration, and nanoparticles conjugated with trastuzumab (Lu177-IONP-Trastuzumab) also decreased cell viability, but the target specificity of trastuzumab was not demonstrated in the HER2 low-expressing cell line, so IC 50 The value was confirmed to be approximately 1098 kBq / mL (29.6 μCi / mL).

[0323]

[0324] - Confirmation of in vivo distribution of antibody-conjugated nanoparticles

[0325] Figure 24 is a drawing showing the results of an experiment to confirm the biodistribution of antibody-conjugated nanoparticles (Lu177-IONP-Trastuzumab) according to one embodiment of the present invention.

[0326] As shown, it shows how much the nanoparticles (100) were distributed in each organ and tumor in the body at one day after injection, and the results of quantitatively measuring the radioactive signal using a γ-counter were confirmed. In addition, the experimental results of Fig. 24 were expressed as the average distribution ratio (% Injected Dose, %ID) on the y-axis at each site on the x-axis.

[0327] As shown, when 100% of the injection dose (24-1) was administered, a small amount of nanoparticles were detected in the blood (24-2), indicating that the nanoparticles were delivered into the body through blood circulation. In addition, it was confirmed that nanoparticles were distributed at a relatively low rate in organs such as the liver (24-4), lungs (24-5), spleen (24-6), and kidneys (24-7). It was found that this was expected to be due to the effect of reducing nonspecific binding of hypervalent iodine to the polymer layer formed during the microwave reaction during the manufacturing process. Accordingly, it was confirmed that the nanoparticles (100) were not selectively accumulated in a specific organ, but were uniformly distributed in the body. In addition, it was confirmed that a very low level of radioactive signal was detected in the femur (24-8).

[0328] In addition, since radioactive pharmaceuticals have a problem that their retention time in the body is long and they can accumulate in the liver and kidneys, which can increase their toxicity in the body, the drug that does not use iodine oxide in the lipid nanoparticle shell (W / O HIBL) and the drug that uses iodine oxide in the lipid nanoparticle shell (W / HIBL) were compared to compare the degree of accumulation in the body. As a result, according to Fig. 27, it was confirmed that the drug that used iodine oxide not only accumulated less in the liver and kidneys but was also washed out faster, so it was found that the composition of iodine oxide in the composition of lipid nanoparticles had a significant improving effect on body toxicity.

[0329]

[0330] - Evaluation of liver and kidney function in experimental animals (mice) administered antibody-conjugated nanoparticles

[0331] FIG. 25 is a diagram showing the results of a serum biochemical analysis performed to evaluate liver function and kidney function in an experimental animal (mouse) administered antibody-conjugated nanoparticles (Lu177-IONP-Trastuzumab) according to one embodiment of the present invention.

[0332] As shown in (a) of Figure 25, in the liver function evaluation, AST (Aspartate Transaminase, 25-1) and ALT (Alanine Transaminase, 25-2) are enzymes that evaluate hepatocyte damage, and both indicators were confirmed to be maintained within the normal range regardless of the radioactivity concentration and time elapsed. This meant that Lu177-IONP-Trastuzumab did not cause hepatocyte damage. In addition, LDH (Lactate Dehydrogenase, 25-3) is an indicator that reflects liver and other tissue damage, and since the LDH (25-3) value was also within the normal range under all measurement conditions, it was confirmed that there was no tissue damage. In addition, ALP (Alkaline Phosphatase, 25-4) is an enzyme that evaluates hepatic bile flow or bone metabolism, and since the data were within the normal range, it was confirmed that there were no signs of hepatic bile flow abnormalities or bone disease. In addition, total bilirubin (25-5) is an indicator of liver metabolic and excretory functions, and the data were within the normal range, indicating that Lu177-IONP-Trastuzumab did not affect liver metabolism. In addition, albumin (25-6) is an indicator of liver protein synthesis function, and all data were within the normal range, indicating that there was no problem with liver synthesis function.

[0333] As shown in (b) of FIG. 25, in the evaluation of renal function, creatinine (Creatinine, 25-7) is a major indicator for evaluating the filtration function of the kidney, and since the data is within the normal range, it was confirmed that Lu177-IONP-Trastuzumab did not affect the renal filtration function. In addition, BUN (Blood Urea Nitrogen, 25-8) is an indicator reflecting the excretion function of the kidney, and since the data is within the normal range, it was confirmed that there was no abnormality in the renal excretion function. That is, it was confirmed that Lu177-IONP-Trastuzumab, which is an example of the nanoparticle-antibody binding complex (200) according to the present invention, maintains safety for the liver and kidneys in vivo and has no toxicity.

[0334]

[0335] - In vivo anticancer efficacy of antibody-conjugated nanoparticles

[0336] Figure 26 is a drawing showing the results of an experiment to confirm the in vivo anticancer efficacy of antibody-conjugated nanoparticles (Lu177-IONP-Trastuzumab) according to one embodiment of the present invention.

[0337] As shown in (a) of Fig. 26, the relative tumor volume (26-1), which is the y-axis of BT-474 (HER2 positive cell line), showed that the tumor size inhibition effect varied depending on the number of administrations of the nanoparticle-antibody conjugate complex (200) in mice with HER2 positive cell line (21-1). In the untreated control group (Vehicle Control, 26-1a), it was confirmed that the tumor size continuously increased and reached more than about 600%. In addition, it was confirmed that in the case of a single administration (40 μCi x 1, 26-1b), the tumor size was inhibited, but increased by about 400%. In addition, in the case of two administrations (40 μCi x 2, 26-1c), it was confirmed that the tumor size was inhibited by about 200%. Additionally, it was confirmed that tumor size was most effectively suppressed when administered three times (40 μCi x 3, 26-1d), and suppressed by approximately 100% or less.

[0338] As shown in (b) of Figure 26, in the body weight change (Body Weight, 26-2) on the y-axis, no change in body weight was observed in any of the treatment groups and the control group, and thus, it was confirmed that the treatment according to the present invention did not have a negative effect on the overall health of the mice.

[0339]

[0340] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. 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 that follow, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

Claims

1. A nanoparticle characterized by comprising an inorganic core formed as a lattice structure compound interface layer containing a radioactive isotope, and further coated with an inorganic shell or organic shell to which a targeting ligand is covalently bonded to the interface layer. The above lattice structure compound is M x X y , wherein M is copper (Cu), silver (Ag) or lutetium (Lu), X is one of fluorine (F), chlorine (Cl), bromine (Br) and iodine (I), x is one of the integers from 1 to 6, and y is one of the integers from 1 to 6. A nanoparticle characterized in that the above-mentioned inorganic shell or organic shell further includes iodine oxide to form an iodine bridge.

2. In claim 1, the radioactive isotope is iodine (I)-123, iodine (I)-125, iodine (I)-131, gallium (Ga)-67, gallium (Ga)-68, copper (Cu)-64, copper (Cu)-67, gold (Au)-198, lead (Pb)-210, nickel (Ni)-63, dysprosium (Dy)-165, radium (Ra)-226, lanthanum (La)-140, rhenium (Re)-186, rhenium (Re)-188, ruthenium (Ru)-82, lutetium (Lu)-177, manganese (Mn)-54, molybdenum (Mo)-99, bismuth (Bi)-213, samarium (Sm)-153, Cesium (Ce)-137, sodium (Na)-24, scandium (Sc)-46, strontium (Sr)-82, strontium (Sr)-85, strontium (Sr)-89, strontium (Sr)-90, americium (Am)-241, zinc (Zn)-65, erbium (Er)-169, uranium (U)-234, uranium (U)-235, uranium (U)-238, silver (Ag)-110m, iridium (Ir)-192, iridium (Ir)-169, iridium (Ir)-177, yttrium (Yt)-169, yttrium (Yt)-177, indium (In)-111, germanium (Ge)-68, Among iron (Fe)-55, cadmium (Cd)-109, calcium (Ca)-47, californium (Cf)-252, cobalt (Co)-57, cobalt (Co)-60, curium (Cm)-244, chromium (Cr)-51, chromium (Cr)-57, krypton (Kr)-81, krypton (Kr)-85, thallium (Tl)-201, thallium (Tl)-204, technetium (Tc)-99m, thorium (Th)-229, thorium (Th)-230, lead (Pd)-103, potassium (K)-42, polonium (Po)-210, promethium (Pm)-147, plutonium (Pu)-238, actinium (Ac)-225, and radium (Ra)-223 A nanoparticle characterized by one of the following:

3. A nanoparticle according to claim 1, wherein the lattice structure compound is characterized in that it forms a radioisotope halide bridge by combining with a radioisotope.

4. In claim 1, the inorganic core includes a metal oxide, and the metal of the metal oxide is characterized in that at least one of iron (Fe), scandium (Sc), titanium (Ti), vanadium (V), chromium (Cr), manganese (Mn), cobalt (Co), nickel (Ni), copper (Cu), yttrium (Y), zirconium (Zr), niobium (Nb), molybdenum (Mo), technetium (Tc), ruthenium (Ru), rhodium (Rh), palladium (Pd), silver (Ag), hafnium (Hf), tantalum (Ta), tungsten (W), rhenium (Re), osmium (Os), iridium (Ir), platinum (Pt), gold (Au), bismuth (Bi), and silicon (Si).

5. In claim 4, the metal oxide is Fe 13 O 19 , Fe3O4 (magnetite), γ-Fe2O3 (magemite) and α-Fe2O3 (hematite), β-Fe2O3 (beta phase), ε-Fe2O3 (epsilon phase), FeO (Wustite), FeO2 (Iron Dioxide), Fe4O5, Fe5O6, Fe5O7, Fe 25 O 32 Nanoparticles characterized by at least one of CuFeO2 (Delafossite).

6. In claim 1, the target ligand is at least one of a peptide, a small molecule, and an antibody, The above peptide is any one of somatostatin, GRP (Gastrin-releasing-peptide), GUL (Glu-Urea-Lys) and PSMA-I&T (Glu-Ureabased ligand), The above small molecule is one of fructose, levan, glucose, adenosine, glycine, glycine, tryptophane, alanine, arginine, lysine, riboflavin, biotin, thiamine, vitamin B12, methotrexate, anisamide, anacardic acid, phenylboric acid, tamoxifen, folate, imatinib, vemurafenib, and sorafenib. The above antibodies are chimeric monoclonal antibodies, humanized monoclonal antibodies, human monoclonal antibodies, antibody fragments, antibody-drug conjugates (ADCs), immunoconjugates, Trastuzumab, Pertuzumab, Ado-Trastuzumab Emtansine, Trastuzumab Deruxtecan, Rituximab, Cetuximab, Nivolumab, Bevacizumab, Atezolizumab, Blinatumomab, Olaratumab, A nanoparticle characterized by being one of daratumumab, elotuzumab, and ipilimumab.

7. A nanoparticle according to claim 1, wherein the inorganic shell or the organic shell is composed of an organic polymer and an inorganic polymer, respectively, and the organic polymer or the inorganic polymer is any one of polyaniline, polyisobutylene, polyethylene, polystyrene, paraffin, graphene, polyvinylpyrrolidone, polyethylene glycol, gelatin, propylene glycol, poly(3,4-ethylenedioxythiophene), poly(methyl methacrylate), and polydimethylsiloxane.

8. A nanoparticle according to claim 1, wherein the lattice structure compound comprises iodine and forms a chemical bond with the radioactive isotope in the interface layer through the iodine element.

9. In claim 1, the nanoparticle is characterized by having a diameter (hydrodynamic size) of 1 nm or more and 200 nm or less.

10. A pharmaceutical composition for treating or preventing cancer, comprising the nanoparticle of claim 1 as an active ingredient.

11. A pharmaceutical composition according to claim 10, characterized in that the cancer disease is one of breast cancer, lung cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, colon cancer, brain tumor, glioblastoma, ovarian cancer, pancreatic cancer, stomach cancer, and esophageal cancer.

12. A pharmaceutical composition according to claim 10, characterized in that the pharmaceutical composition is a radiopharmaceutical for targeted therapy.

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