Surface-modified albumin nano-platform, method for preparing same, and composition for diagnosing or treating disease using same

Abstract

The present invention relates to a surface-modified albumin nano-platform, a method for preparing same, and a composition for diagnosing or treating a disease using same. The surface-modified albumin nano-platform according to the present invention can reduce ingestion by the reticuloendothelial system even when an excess amount of click-functional groups and various delivery substances are conjugated to albumin, by inactivating a click reaction functional group to which a delivery substance on the surface of albumin is not bound, so that the in vivo half-life is greatly increased, and a target molecule or an active substance can be effectively delivered to a target tissue or cell, leading to excellent therapeutic and diagnostic effects.

Description

Surface-modified albumin nanoplatform, method for preparing the same, and composition for diagnosing or treating diseases using the same

[0001] The present invention relates to a surface-modified albumin nanoplatform, a method for producing the same, and a composition for diagnosing or treating a disease using the same. More specifically, the present invention provides an albumin nanoplatform with an increased half-life in the body by masking and inactivating a click functional group to which a delivery substance is not bound on the surface of the albumin nanoplatform, thereby reducing uptake by the reticuloendothelial system, and a method for producing the same, and a composition for diagnosing or treating a disease using the same.

[0002] Albumin is a highly stable protein that circulates in the blood for more than 20 days. It is used as a drug delivery system to enhance the in vivo stability of various drugs by chemically binding existing small-molecule chemical drugs, proteins, or shapes to albumin. These albumin-based drug delivery systems are utilized in various fields of medicine and pharmacy for diagnosis and treatment. Representative studies have focused on chemically binding hydrophobic anticancer drugs such as doxorubicin and methotrexate (MTX) to albumin to enhance cancer targeting and blood stability.

[0003] In addition, active research has been conducted on manufacturing nanoparticles using albumin. Since albumin is a biodegradable substance that is non-toxic, non-immunogenic, and decomposable in the body, nanoparticles formed from albumin are very useful as drug delivery vehicles.

[0004] However, many studies have shown that nanoparticle-based drug delivery methods using various substances such as albumin, liposomes, or antibodies show a short drug retention time in the body due to rapid uptake into the reticuloendothelial system, including the liver or spleen, when injected into the body, which limits their use. To solve this problem, there is a method to increase the amount of nanoparticle-based drug delivery vehicles used, but there are toxicity issues due to the physical properties of nanoparticles, and problems with immune responses have always been raised. To date, technological development to solve the short drug retention time of nanoparticles in the body is insufficient.

[0005] Korean Patent No. 10-1043407 discloses a technology for forming a drug delivery complex by combining hydrophilic albumin and hydrophobic bile acid. However, the protein complex forms self-assemblies in a hydrophilic environment to deliver drugs in the form of nanoparticles, and is specifically intended for the delivery of hydrophobic drugs. The effect of increasing the half-life in the body is not disclosed.

[0006] As the importance of albumin nanoparticles as drug delivery vehicles is receiving attention, there is a need to develop a technology to delay the uptake of albumin nanoparticles from the reticuloendothelial system and increase the drug retention time in the body.

[0007] The purpose of the present invention is to provide a surface-modified albumin nanoplatform to increase the half-life in the body.

[0008] Another object of the present invention is to provide a method for manufacturing a surface-modified albumin nanoplatform with increased in vivo half-life.

[0009] Another object of the present invention is to provide a composition for diagnosing or treating a disease comprising a surface-modified albumin nanoplatform with increased half-life in the body.

[0010] The present invention provides a surface-modified albumin nanoplatform obtained by a click chemistry reaction between albumin having an azide (N3) or cyclooctyne functional group bound thereto and a carrier having an azide (N3) or cyclooctyne functional group bound thereto, wherein when the albumin is bound to the azide functional group, the carrier is bound to the cyclooctyne functional group, and when the albumin is bound to the cyclooctyne functional group, the carrier is bound to the azide functional group, and wherein at least one of the azide or cyclooctyne functional groups bound to the albumin to which the carrier is not bound is inactivated.

[0011] In the present invention, the number of azide or cyclooctane functional groups introduced into the albumin may be 1 to 30.

[0012] In the present invention, the inactivation can be performed by binding a masking material having a substituent selected from the group consisting of an amino group (-NH2), a carboxyl group (-COOH), and a hydroxyl group (-OH) to an azide or cyclooctane functional group bound to albumin.

[0013] In the present invention, the bonding of the masking material can be performed by a click chemical reaction between the masking material having an azide or cyclooctane functional group bonded to the albumin having the delivery material bonded to it.

[0014] In the present invention, when the albumin is combined with an azide functional group, the masking material may be combined with a cyclooctane functional group, and when the albumin is combined with a cyclooctane functional group, the masking material may be combined with an azide functional group.

[0015] In the present invention, the delivery substance may be at least one selected from the group consisting of a sugar compound, folic acid or a derivative thereof, RGD (arginyl-glycyl-aspartic acid) or a derivative thereof, an active substance, a radioactive isotope, and a fluorescent substance.

[0016] In the present invention, the sugar compound may be at least one selected from the group consisting of a glucosyl group, a mannosyl group, and a galactosyl group.

[0017] In the present invention, the radioactive isotope is 3 H, 11 C, 18 F, 14 Cl, 32 P, 35 S, 36 Cl, 45 Ca, 51 Cr, 57 Co, 58 Co, 59 F, 64 Cu, 67 Ga, 68 Ga, 89 Zr, 90 Y, 99 Mo, 99 mTc, 111 In, 131 I, 125 I, 124 I, 123 I, 186 Re, 188 Re, 225 Ac, 212 Pb, 117 mSn, and 177 It may be one or more selected from the group consisting of Lu.

[0018] In the present invention, the radioactive isotope is represented by a chelating agent, and the chelating agent is NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DFO (3-[6,17-dihyroxy-7,10,18,21-tetraoxo-27-[N-acetylhydroxylamino)-6,11,17,22-tetraazaheptaeicosane]thiourea), DTPA (diethylenetriaminepentaacetic acid), N2S2 (diaminedithiol), p-SCN-Bn-NOTA (2-(4'-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid), NODAGA (1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid), p-SCN-Bn-DOTA (2-(4'-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), p-SCN-Bn-DTPA (2-(4-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid), p-SCN-Bn-DFO It may be at least one selected from the group consisting of (1-(4-Isothiocyanatophenyl)-3-[6,17-dihyroxy-7,10,18,21-tetraoxo-27-[Nacetylhydroxylamino)-6,11,17,22-tetraazaheptaeicosane]thiourea) and HYNIC (hydrazinonicotinic acid).

[0019] In the present invention, the fluorescent material is FNR (Ferrodoxin NADP(+) reductase), cyanine-based fluorescent material, TAMRA (tetramethylrhodamine-5-maleimide), Flamma ® It may be at least one selected from the group consisting of fluorescent substances and ICG (indocyanine green).

[0020] The present invention also comprises the steps of: (1) preparing an albumin solution having an azide (N3) or cyclooctyne functional group attached thereto; (2) mixing a solution containing a carrier having a cyclooctyne functional group or an azide (N3) functional group attached thereto with the solution prepared in step (1) and subjecting the solution to a copper-free click chemistry reaction; And (3) a step of mixing a solution containing a masking material having a cyclooctane functional group or an azide (N3) functional group bonded to an albumin solution to which a carrier is bonded and performing a copper-free click chemistry reaction, wherein when the albumin is bonded to an azide functional group, the carrier and masking material are bonded to a cyclooctyne functional group, and when the albumin is bonded to a cyclooctyne functional group, the carrier and masking material are bonded to an azide functional group, thereby providing a method for manufacturing a surface-modified albumin nanoplatform.

[0021] In the method of the present invention, in step (3), the molar ratio of albumin and masking material may be 1:10 to 1:100.

[0022] The present invention also provides a composition for diagnosing or treating a disease comprising the surface-modified albumin nanoplatform.

[0023] In the present invention, the disease may be selected from acute nephritis, periodontitis, sarcopenia, pancreatic cancer, head and neck cancer, endometrial cancer, bone cancer, breast cancer, central or peripheral nervous system cancer, digestive cancer, germ cell cancer, adenocarcinoma, blood cancer, renal-urinary tract cancer, liver cancer, lung cancer, pleural cancer, prostate cancer, sarcoma, and skin cancer.

[0024] The surface-modified albumin nanoplatform according to the present invention can reduce uptake by the reticuloendothelial system even when excessive click functional groups and various transporters are bound to albumin by inactivating the click reaction functional groups on the albumin surface to which the transporter is not bound, thereby greatly increasing the half-life in the body, thereby effectively delivering target molecules or active substances to target tissues or cells, thereby achieving excellent therapeutic and diagnostic effects.

[0025] Figure 1 shows the results of confirming the residual amount in blood by confirming the fluorescence expression levels of non-masking and masked albumin according to one embodiment of the present invention.

[0026] Figure 2 is a diagram illustrating an unmasked mannosylated albumin according to one embodiment of the present invention. 64 Cu-Alb-Man) and masked mannosylated albumin ( 64 This is a PET image result confirming fluorescence expression in blood, liver tissue, and cancer tissue in vivo after administration of Cu-Alb-Man-blocking) to a tumor animal model.

[0027] FIG. 3a is a graph showing the expression level in blood among the PET images of FIG. 2 according to one embodiment of the present invention.

[0028] FIG. 3b is a graph showing the level of fluorescence expression in liver tissue among the PET images of FIG. 2 according to one embodiment of the present invention.

[0029] FIG. 4 is a graph showing the level of fluorescence expression within a tumor in the PET image of FIG. 2 according to one embodiment of the present invention.

[0030] Hereinafter, specific implementations of the present invention will be described in more detail. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0031] Throughout the specification, whenever a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise stated.

[0032]

[0033] The present invention provides a surface-modified albumin-based nanoplatform and a method for manufacturing the same to increase the half-life in the body.

[0034] In a previous study, the present inventors proposed an albumin nanoplatform having a click-activated surface functional group that reacts with the amine functional group on the albumin surface (Patent No. 10-1879682, Albumin-Based Disease Tracking, Diagnosis, or Treatment Nanoplatform). The previous study suggested that albumin and the click functional group react at a molar ratio of 1:1 to 1:12. This was because reactions using molar ratios higher than this resulted in rapid uptake into the reticuloendothelial system, significantly limiting the usability of the nanoplatform.

[0035] However, in order to introduce click-activated functional groups into various target substances or drugs, it is more effective to introduce more than 9 click-activated functional groups onto the albumin surface by reacting the click reactor at a molar ratio of 1:12 or more.

[0036] Therefore, the present invention aims to provide an albumin nanoplatform that combines a number of click-reactive functional groups on the surface of albumin while reducing the uptake rate in the liver and significantly improving the half-life in the body.

[0037] Specifically, in the present invention, in order to increase the in vivo half-life of an albumin nanoplatform having a click-reactive functional group bound to the surface, a masking material is bound to the click-reactive functional group on the surface of the albumin nanoplatform to inactivate it, thereby reducing uptake by the reticuloendothelial system, thereby providing an albumin nanoplatform having a significantly increased in vivo half-life.

[0038]

[0039] In this respect, the present invention provides an albumin nanoplatform obtained by a click chemistry reaction of albumin having an azide (N3) or cyclooctyne functional group bound thereto and a carrier having an azide (N3) or cyclooctyne functional group bound thereto, wherein when the albumin is bound to the azide functional group, the carrier is bound to the cyclooctyne functional group, and when the albumin is bound to the cyclooctyne functional group, the carrier is bound to the azide functional group, and wherein at least one of the azide or cyclooctyne functional groups of the albumin nanoplatform to which the carrier is not bound is inactivated.

[0040] Albumin is a protein that constitutes the basic building blocks of cells. It is found in high concentrations in the blood and is produced in the liver. Albumin has the lowest molecular weight among simple proteins found in nature. Serum albumin in the blood maintains and restores plasma volume, preventing shock from excessive bleeding and being used in surgery and burn treatment. It is also known to possess oxygen-carrying properties similar to hemoglobin.

[0041] The albumin described above may include any albumin that can be formulated, but is preferably derived from human plasma or recombinant human serum albumin produced through genetic engineering, but is not limited thereto. Genetic information regarding the albumin of the present invention can be obtained from known databases such as NCBI GenBank.

[0042] The number of amino groups (-NH2) exposed on the surface of albumin may be 15 to 35.

[0043] In a specific embodiment of the present invention, a nanoplatform capable of delivering a drug or a fluorescent substance to a target tissue in a living body based on biocompatible albumin to treat or diagnose a disease was manufactured, and a representative human serum albumin (HSA) was used, and a cyclooctane or azide (N3) group, which is one functional group of click chemistry, was introduced to the surface of human serum albumin under reaction conditions that minimized the denaturation of HSA.

[0044] In the present invention, azide (N3) is a reactive group composed of three nitrogen atoms, has high reactivity, and is known to play a role as an electron donor in the 1,3-dipolar cycloaddition reaction, which is a type of Cu-free click chemistry, to form a triaza-5-membered ring.

[0045] The cyclooctyne functional group is an eight-membered aliphatic ring containing a triple bond under ring strain. It is known to act as an electron acceptor, particularly in the 1,3-dipolar cycloaddition reaction, a type of Cu-free click chemistry, to form a triaza-5-membered ring. The structural characteristics of cyclooctyne, namely the triple bond structure under ring strain, enable click chemistry even without a Cu(I) catalyst.

[0046] The cyclooctyn functional group is 4-cyclooctyn-1-yl( ), 3-cyclooctyn-1-yl( ), 2-cyclooctyn-1-yl( ), Monofluorinated cyclooctyne (MOFO) ( ), Difluorinated cyclooctyne (DIFO) ( ), Dimethoxyazacyclooctyne (DIMAC) ( ), Dibenzocyclooctyne(DIBO)( ), Azadibenzocyclooctyne (ADIBO) group ( ) and biarylazacyclooctynone (BARAC) ( ) may be one or more selected from the group consisting of, but is not necessarily limited thereto.

[0047] Albumin having an azide (N3) or cyclooctyne functional group attached can be obtained by the steps of (a) dissolving albumin in phosphate-buffered saline (PBS), (b) dissolving azide-NHS or cyclooctyne-NHS in DMSO, and (c) mixing the obtained solutions and reacting them at 20 to 37°C for 30 minutes to 1 hour.

[0048] (a) In step, the phosphate buffer solution (PBS) may have a pH of 6.8 to 7.6, and preferably a pH of 7.0 to 7.4.

[0049] (b) The amount of DMSO used to prepare the azide-NHS solution or the cyclooctane-NHS solution in step may be 2% (v / v) or less of the total reaction solution.

[0050] (c) The mixing molar ratio of albumin and azide-NHS or cyclooctane-NHS in step may be 1:1 to 1:30.

[0051] (c) When mixing an albumin solution and an azide-NHS solution in step (c), the functional group bound to the albumin may be an azide functional group, and when mixing an albumin solution and a cyclooctane-NHS solution in step (c), the functional group bound to the albumin may be a cyclooctane functional group.

[0052] In one embodiment of the present invention, a human serum albumin (HSA) solution and an ADIBO-NHS solution were mixed and reacted at 37°C for 30 minutes to produce HSA-ADIBO.

[0053] In the present invention, the number of click reaction functional groups (azide or cyclooctane functional groups) introduced to the albumin surface by this method is preferably 1 to 30, and more preferably 9 to 20.

[0054] In the inventor's prior art, if the number of click-reactive functional groups exceeds 10, the drug may be immediately taken up by the liver upon injection, thereby limiting uptake to other target disease sites. Therefore, the number of click-reactive functional groups was limited. However, according to the present invention, liver uptake is limited by inactivating the click-reactive functional groups to which the transducer is not bound. Therefore, by introducing a larger number of click-reactive functional groups, a wider variety of transducers can be bound, while also extending the duration of retention in the body.

[0055] The number of click-reactive functional groups introduced on the albumin surface can be controlled by the reaction ratio of albumin and azide-NHS or cyclooctane-NHS.

[0056] In the present invention, a masking material can be reacted with a click reaction functional group bound to the albumin surface to inactivate it. The masking material can be a material capable of inactivating the click reaction functional group, and is specifically a material having a substituent selected from the group consisting of an amino group (-NH2), a carboxyl group (-COOH), and a hydroxyl group (-OH). In particular, one having an amino group is most preferable in terms of enabling the click reaction functional group to be recognized by the surface of albumin. As a compound containing the amino group and carboxyl group, for example, lysine can be used, and as a compound containing a hydroxyl group, for example, polyethylene glycol (PEG) can be used.

[0057] The above masking material reacts with a click-reactive functional group on the surface of the albumin nanoplatform that does not bind to the delivery substance, thereby deactivating it and returning the albumin nanoplatform to the state of the albumin surface having the existing amino or carboxyl functional group, thereby reducing uptake by the reticuloendothelial system without affecting the targeting or therapeutic ability of the delivery substance, thereby significantly increasing the half-life of the albumin nanoplatform in the body.

[0058] In one embodiment of the present invention, as a result of confirming the distribution of albumin in the body according to masking, in mice injected with non-masking albumin nanoplatform, rapid uptake into the liver was observed as DOF ​​increased, whereas in mice administered with albumin nanoplatform masked with an amine group, the fluorescence level was maintained for a long time even at a high DOF, confirming that the albumin nanoplatform surface-modified with a masking material decreased uptake into the liver and increased the residual amount in the blood, thereby increasing the circulation half-life.

[0059] In the present invention, the albumin nanoplatform is obtained by mixing a solution containing albumin having an azide or cyclooctane functional group bonded thereto and a solution containing a carrier having an azide or cyclooctane functional group bonded thereto, and performing a click chemistry reaction, wherein the click chemistry reaction may be a copper-free click chemistry reaction.

[0060] In one embodiment of the present invention, the azide functional group used as the click chemistry functional group is an electron donor, and the cyclooctane functional group is an electron acceptor. Therefore, when the functional group bonded to albumin is an azide functional group, the functional group bonded to the carrier is preferably a cyclooctane functional group, and when the functional group bonded to albumin is a cyclooctane functional group, the functional group bonded to the carrier is preferably an azide functional group.

[0061] Next, a solution containing a masking material with an azide or cyclooctane functional group is mixed with the albumin nanoplatform solution to which the above-mentioned carrier is bound, and a click chemical reaction is performed to obtain a surface-modified albumin nanoplatform. At this time, the azide or cyclooctane functional group bound to the masking material is preferably the same as the functional group bound to the carrier.

[0062] For example, as azide-bonded masking substances, HL-Lys(N3)-OH, HL-Aha-OH, HL-Lys(N3)-OH*HCl, HL-Aha-OH*HCl, HL-Phe(4-N3)-OH, 3-azido-1-propanol, 3-azidopropionic acid, etc. can be used, and the above compounds can include PEG as a linker.

[0063] In this respect, the surface-modified albumin nanoplatform of the present invention can be manufactured by (1) preparing an albumin solution having an azide (N3) or cyclooctyne functional group attached thereto; (2) mixing a solution containing a carrier having a cyclooctane functional group or an azide (N3) functional group attached thereto with the solution prepared in step (1) and subjecting the solution to a copper-free click chemistry reaction; and (3) mixing a solution containing a masking material having a cyclooctane functional group or an azide (N3) functional group attached thereto with the albumin solution having the carrier group attached thereto and subjecting the solution to a copper-free click chemistry reaction.

[0064] At this time, when using a solution in which an azide (N3) group is bonded to an albumin monomer in step (1), it is preferable to react with a solution containing a carrier and a masking material bonded to a cyclooctane group in steps (2) and (3), and when using a solution in which an albumin monomer is bonded to a cyclooctane functional group in step (1), it is preferable to react with a solution containing a carrier and a masking material bonded to an azide (N3) functional group in steps (2) and (3).

[0065] In the above step (3), the albumin and masking material to which the transfer agent is bound can be mixed in a molar ratio of 1:10 to 1:100, preferably 1:20 to 1:60. This can be adjusted depending on the number of functional groups to which the transfer agent is not bound among the click reaction functional groups bound to the albumin surface, and it is preferable to use an excess of the masking material in the sense that all of the click reaction functional groups are inactivated as much as possible. For example, the molar ratio of the albumin and the masking material can be 1:30 to 1:50. When albumin and the masking material are reacted in the above molar ratio range, all of the click reaction functional groups to which the transfer agent is not bound on the albumin surface can be inactivated at a rapid rate of 2 to 6 hours.

[0066] The above step (3) reaction may be performed under pH conditions of 6 to 7 to maintain albumin stability. If the pH is higher than the above pH range, thermal denaturation of albumin may occur, and if the pH is lower than the above pH range, decomposition and oxidative damage due to acidification may occur.

[0067] In addition, the reaction temperature of step (3) can be performed at 0 to 60°C, but it is preferable to perform it at room temperature in order to induce a faster binding reaction between the click reaction functional group and the masking material while maintaining the stability of albumin.

[0068] The albumin nanoplatform of the present invention possesses multiple click-responsive functional groups, enabling binding to various carriers. The binding agent capable of binding to the albumin nanoplatform of the present invention may be a single agent or a combination of various binding agents.

[0069] When the delivery material bound to the albumin nanoplatform is a combination of two or more materials, the combination of two or more materials may be bound to the albumin nanoplatform simultaneously, or each material may be bound sequentially, but is not limited thereto.

[0070] In the present invention, the delivery material means a material that is delivered into a living body by binding to the albumin nanoplatform, and may be selected from the group consisting of target molecules, active substances, radioactive isotopes, fluorescent substances, and combinations thereof.

[0071] The term "target molecule" of the present invention refers to a molecule that targets a specific environment in a living body, and specifically refers to a molecule that targets a substance existing in a specific environment in a living body and guides the albumin nanoplatform of the present invention to the corresponding specific environment. The target molecule of the present invention can guide the albumin nanoplatform of the present invention to a specific environment, or confirm the presence or absence of a specific environment, the location of the specific environment, the degree of the specific environment, etc. In the present invention, the specific environment in a living body targeted by the target molecule may be a tissue-specific specific environment or a disease-specific specific environment. In other words, the target molecule may target a substance existing tissue-specifically or a disease-specific substance. A tissue-specific substance may be a protein, RNA, a compound accumulated or produced, etc. existing tissue-specifically, and a disease-specific substance may be a protein, RNA, a compound accumulated or produced, etc. existing depending on the type of disease.

[0072] In particular, in the present invention, the target molecule may be a sugar compound, folate or a derivative thereof that targets a folate receptor present in large numbers in cancer tissue, or RGD (arginyl-glycyl-aspartic acid) or a derivative thereof.

[0073] In one embodiment of the present invention, the transmitter may be a sugar compound, and the sugar compound may be a mannosyl group, a galactosyl group, or a glucosyl group. The structures of the mannosyl group, the galactosyl group, and the glucosyl group are as shown in Formulas 1 to 3 below, respectively.

[0074]

[0075] [Formula 1]

[0076]

[0077] [Formula 2]

[0078]

[0079] [Formula 3]

[0080]

[0081]

[0082] Due to this type of sugar compound delivery material, the albumin nanoplatform, which is one embodiment of the present invention, has a targeting ability for macrophages, and this has been described in detail in the inventor's prior research, Patent No. 10-2366189.

[0083] Specifically, an albumin nanoplatform containing a mannosyl group or galactosyl group as a delivery agent has targeting ability for macrophages, thereby enabling drug delivery to macrophages, and as a result, can function as a platform for preemptive diagnosis and treatment of metastatic cancer.

[0084] Mannose receptors are known to be abundant on cells involved in defense mechanisms, with Kupffer cells being a prime example of such immune cells. When the transmitter is mannosyl, it targets Kupffer cells, so it typically acts in the liver, but it can also affect the blood, muscles, spleen, and lungs.

[0085] In addition, when the transmitter is a galactosyl group, it can exhibit hepatobiliary excretion through the gall bladder. In addition, the albumin-based nanoplatform of the present invention, in which a glucosyl group is bound as a transmitter, has a targeting ability for M1 type macrophages, and through this, it can be selectively taken up by inflammatory macrophages in damaged tissues, suppress the inflammatory response and ROS production induced by inflammatory macrophages, restore the function of damaged mitochondria, and suppress cell death in damaged tissues, so that it can be used as a nanoplatform that can prevent or treat progression to serious diseases.

[0086] In the present invention, the albumin nanoplatform may contain 4 to 8 sugar compounds, more preferably 5 to 7 sugar compounds.

[0087] In the present invention, folic acid can be combined as the above-mentioned transmitting agent.

[0088] Folic acid is a vitamin, also known as vitamin B9 or vitamin M. Folic acid binds specifically to the folate receptor. The folate receptor is a tumor-associated glycosylphosphatidylinositol anchor protein that allows bound folate or folate-conjugated substances to be uptaken through receptor-mediated endocytosis.

[0089] In the present invention, the delivery molecule RGD is a tripeptide composed of three amino acids—arginine (L-arginine), glycine (glycine), and aspartic acid (L-aspartic acid). It was first discovered in the cell adhesion region of fibronectin. Because it possesses a strong affinity for integrin receptors, which are primarily expressed on the endothelial cell layer of tumor blood vessels, it is widely used as a universal tool in research on drug-targeted therapy and simple reagents.

[0090] The term "active substance" of the present invention may be a natural product, vitamin, active agent, biological agent, compound, amino acid, protein, peptide, nucleotide, nucleic acid, polypeptide, polynucleotide and analogs, homologues and derivatives thereof, and in particular, an anti-obesity drug, a central nervous system stimulant, a carotenoid, a corticosteroid, an elastase inhibitor, an antifungal agent, an anti-tumor agent, an antiemetic, an analgesic, a cardiovascular agent, an anti-inflammatory agent, an anthelmintic agent, an antiarrhythmic agent, an antibiotic, an anticoagulant, an antidepressant, an antidiabetic agent, an antiepileptic agent, an antihistamine, an antihypertensive agent, an antimuscarinic agent, an antimycobacterial agent, an immunosuppressant, an antithyroid agent, an antiviral agent, an anxiolytic agent, a sedative, an astringent, an alpha-adrenergic receptor blocker, a beta-adrenergic receptor blocker, a blood product, a cardiac inotropic agent, a contrast medium, a cough suppressant, a diagnostic reagent, a diagnostic It may be selected from imaging agents, diuretics, dopaminergic agents, hemostatic agents, immunomodulators, lipid-regulating agents, muscle relaxants, parasympathomimetics, anti-allergic agents, appetite suppressants, sympathomimetics, thyroid agents, vasodilators, and combinations thereof.

[0091] In the present invention, the radioactive isotope as a carrier material can be labeled by a chelating agent.

[0092] Radioisotopes are elements with the same atomic number but different atomic masses. Among these isotopes, those that are radioactive are called radioisotopes. These radioisotopes can be used as valuable markers for diagnosing diseases or assessing pharmacodynamics by utilizing their characteristic ability to attenuate radioactivity by emitting gamma rays or other subatomic particles.

[0093] In the present invention, any radioactive isotope known in the art that can be used as a marker may be used without limitation. 3 H, 11 C, 18 F, 14 Cl, 32 P, 35 S, 36 Cl, 45 Ca, 51Cr, 57 Co, 58 Co, 59 F, 64 Cu, 67 Ga, 68 Ga, 89 Zr, 90 Y, 99 Mo, 99 mTc, 111 In, 131 I, 125 I, 124 I, 123 I, 186 Re, 188 Re, 225 Ac, 212 Pb, 117 mSn, and 177 It may be at least one selected from the group consisting of Lu, and preferably 11 C, 18 F, 64 Cu, 67 Ga, 68 Ga, 89 Zr, 99 mTc, 111 In and 123 It may be I, but is not necessarily limited to this.

[0094] Chelating agents are those that bind radioactive isotopes to albumin, for example, NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DFO (3-[6,17-dihyroxy-7,10,18,21-tetraoxo-27-[N-acetylhydroxylamino)-6,11,17,22-tetraazaheptaeicosane]thiourea), DTPA (diethylenetriaminepentaacetic acid), N2S2 (diaminedithiol), p-SCN-Bn-NOTA (2-(4'-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid), NODAGA (1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid), p-SCN-Bn-DOTA (2-(4'-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), p-SCN-Bn-DTPA (2-(4-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid), p-SCN-Bn-DFO It may be at least one selected from the group consisting of (1-(4-Isothiocyanatophenyl)-3-[6,17-dihyroxy-7,10,18,21-tetraoxo-27-[Nacetylhydroxylamino)-6,11,17,22-tetraazaheptaeicosane]thiourea) and HYNIC (hydrazinonicotinic acid), but is not necessarily limited thereto.

[0095] In the present invention, the transmitting material may further include a fluorescent material.

[0096] A fluorescent material refers to a material that emits a specific wavelength of visible light for a specific wavelength, and in the present invention, all fluorescent materials that can be combined with a nanoplatform for albumin-based macrophage targeting and used to identify the location of the nanoplatform are included.

[0097] Specifically, in the present invention, fluorescent materials that can be used as markers are those known in the art without limitation, and include, but are not limited to, rhodamine-based fluorescent materials including rhodamine, TAMRA, etc.; fluorescein-based fluorescent materials including fluorescein isothiocyanate (FITC) and fluorescein amidite (FAM), etc.; bodipy-based fluorescent materials (boron-dipyrromethene); alexa fluor-based fluorescent materials; and cyanine-based fluorescent materials including Cy3, Cy5, Cy7, and indocyanine green.

[0098]

[0099] The surface-modified albumin nanoplatform according to the present invention has a long half-life in the body, so that it can be utilized as a composition for diagnosing or treating diseases by allowing target molecules and / or active substances to be maintained in the body for a long period of time.

[0100] The above disease is not limited to any disease that can be diagnosed or treated by the delivery material coupled to the albumin nanoplatform of the present invention, and may be, for example, an inflammatory disease such as acute nephritis, periodontitis, sarcopenia, etc., or a cancer such as pancreatic cancer, head and neck cancer, endometrial cancer, bone cancer, breast cancer, central / peripheral nervous system cancer, digestive cancer, germ cell cancer, adenocarcinoma, blood cancer, renal-urinary tract cancer, liver cancer, lung / pleural cancer, prostate cancer, sarcoma, skin cancer, etc.

[0101]

[0102] In this way, the albumin nanoplatform manufactured by the method of the present invention can reduce uptake by the reticuloendothelial system even when excessive click functional groups and various transporters are bound to albumin by inactivating the click functional groups on the albumin surface to which the transporter is not bound, and accordingly, target molecules or active substances can be effectively delivered to target tissues or cells through increased half-life in the body, thereby obtaining excellent therapeutic and diagnostic effects.

[0103]

[0104] Example

[0105]

[0106] The present invention is described in more detail through the following examples. However, these examples are provided to illustrate some experimental methods and configurations for the purpose of illustratively explaining the present invention, and the scope of the present invention is not limited to these examples.

[0107]

[0108] Manufacturing Example 1: Manufacturing of Albumin-Based Nanoplatform

[0109]

[0110] 1-1. Preparation of albumin with ADIBO functional group

[0111]

[0112] Human serum albumin (HSA) was dissolved in PBS at a concentration of 5 mg / 0.5 mL, and 0.5 mL of albumin was dispensed into Ependorf (EP) tubes. ADIBO-NHS dissolved in DMSO was added at molar ratios of 1:3, 1:6, 1:12, 1:13, 1:16, 1:20, and 1:24, and vortexed to disperse rapidly. After sonication for 1 minute, the mixture was stirred at room temperature for 4 hours to react, and purified using a 30 kDa centrifugal filter.

[0113] The purified albumin was transferred to a new EP tube at a volume of 0.5 mL each, and the number of ADIBO functional groups introduced into HSA-ADIBO was quantified using UV-Vis spectrophotometric method (A280, A309) and MALDI-TOF, and is shown in Table 1.

[0114]

[0115]

[0116]

[0117] It was confirmed that approximately 16 ADIBOs could be combined when mixed at a molar ratio of up to 1:24.

[0118]

[0119] 1-2. Albumin Nanoplatform Masking

[0120]

[0121] Among the albumin nanoplatforms prepared as in Manufacturing Example 1-1, sample numbers 2, 4, 6 and 8 (ADIBO numbers 2, 10, 12 and 16) 64 After Cu labeling, masking was performed with N3-NH2.

[0122] First, on the ADIBO functional group on the albumin surface 64 To label Cu, 64 The vial containing Cu was dried under N2 gas in a hood for 30 minutes. Then, 200 μL of 1 M sodium acetate buffer (pH 5) was added to the vial to adjust the pH to 5, and NOTA-N3 (1 mg / mL in DW, 10 μL) was added and heated at 70°C for 5 minutes.

[0123] Thin layer chromatography (TLC) was performed using ITLC-SG paper to measure the radiolabeling efficiency (Radio-TLC, 0.1 M citric acid as the mobile phase). After confirming the radiolabeling efficiency, a solution of radiolabeled NOTA-N3 ([ 6410 μL (9 nmole, 0.5 mCi) of Cu]Cu-NOTA-N3, 10 mCi / 200 μL) was added.

[0124] Alb was added to PBS (500 μg / 0.5 mL, 15 nmole / 0.5 mL) and reacted at room temperature (RT) for 30 minutes, and finally unlabeled 64 Cu, [ 64 The Rf value of Cu] was confirmed. Radio-TLC was performed to confirm the production of radiolabeled Alb-ADIBO, and the result was unlabeled. 64 It was confirmed that Cu was absent.

[0125] Next, 50 μL of 3-Azido-1-propanamine dissolved in distilled water at a concentration of 100 mg / 88 μL was added. 64 After adding Cu-labeled Alb-ADIBO (2 mg / 0.8 ml), blocking was performed at 4°C and centrifugation was performed at 10,000 rpm for 3 minutes at 4°C to obtain masked albumin. The masking agent reacted at a molar ratio of approximately 40 times that of albumin, and based on the 16 ADIBO functional groups bound to albumin, it was confirmed that ADIBO:N3-NH2 reacted at a ratio of 1:25 or higher.

[0126]

[0127] Experimental Example 1: Confirmation of Albumin Distribution in the Body According to Masking

[0128]

[0129] As in Manufacturing Example 1, the isotope-labeled albumin nanoplatform was masked and administered into the tail vein of a normal mouse, and the fluorescence distribution was confirmed at different time points (0, 3, 8, and 24 hours) using PET (positron emission tomography) equipment, and is shown in Figure 1.

[0130] Referring to Figure 1, it was confirmed that in mice injected with the non-masking albumin nanoplatform, rapid uptake into the liver occurred as the DOF increased.

[0131] On the other hand, in mice administered with the N3-NH2 masked albumin nanoplatform, it was confirmed that fluorescence levels similar to DOF 10 were observed even at DOF 12 and DOF 16, which are higher than DOF 10.

[0132] Through this, it was confirmed that the albumin nanoplatform masked with N3-NH2 had a reduced liver uptake and a higher residual amount in the blood, which significantly increased the blood circulation half-life.

[0133]

[0134] Manufacturing Example 2: Manufacturing of Masked Glycated Albumin Nanoplatform

[0135]

[0136] HSA-ADIBO prepared as in Manufacturing Example 1-1 was dissolved in PBS, and Man-N3 (1-O-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-alpha-D-mannopyranoside) was mixed at a molar ratio of 1:8 for HSA-ADIBO:Man-N3, reacted at 37°C for 1 hour, and purified using a centrifugal filter tube to prepare albumin (Alb-Man) with a mannosyl group attached.

[0137] Afterwards, the albumin with the mannosyl group attached by the method of manufacturing example 1-2 64 Surface-modified mannosylated albumin nanoplatforms labeled with Cu and masked with N3-NH2 ( 64 (Cu-Alb-Man-blocking) was obtained.

[0138]

[0139] Experimental Example 2: Determination of the Residual Amount of Masked Albumin Nanoplatform in Blood

[0140]

[0141] The blood residual amount and liver uptake level of the masked albumin nanoplatform were confirmed.

[0142] Surface-modified albumin nanoplatform manufactured by the method of Manufacturing Example 2 ( 64Cu-Alb-Man-Blocking) and unmasked albumin nanoplatform control ( 64 Cu-Alb-Man) was administered to the tail vein of each tumor mouse model. Afterwards, the level of fluorescence expression by the isotope was confirmed through PET imaging of the tumor animal model, and is shown in Figure 2.

[0143] Referring to graph Fig. 3a showing the level of fluorescence expression in blood from the PET image of Fig. 2 and graph Fig. 3b showing the level of fluorescence expression in liver tissue, the residual amounts in blood of the masked albumin nanoplatform and the non-masked control group were at similar levels, but in the case of the non-masked control group, it was confirmed that the residual amount in blood decreased rapidly due to liver uptake. On the other hand, it was confirmed that the time taken for the masked albumin nanoplatform to be uptaken into the liver was delayed.

[0144]

[0145] Experimental Example 3: Confirmation of the Targeting Ability of a Masked Glycated Albumin Nanoplatform

[0146]

[0147] Masked mannosylated albumin nanoplatform of Experimental Example 2 ( 64 Cu-Alb-Man-Blocking) or non-masking control ( 64 The level of isotope fluorescence expression in tumor tissue was confirmed through PET imaging of tumor animal models administered with each of Cu-Alb-Man, to determine whether masking affected the tumor targeting ability of the albumin nanoplatform.

[0148] Referring to Figure 4, which confirms the targeting ability of unmasked or masked mannosylated albumin, it was confirmed that the masked albumin nanoplatform targets tumors at a similar level to the unmasked control group, thereby confirming that masking does not affect the tumor targeting ability of the albumin nanoplatform.

[0149]

[0150] In summary of the above results, the masking method of the present invention, which modifies the surface of an albumin nanoplatform into which an excessive amount of ADIBO groups are introduced, can improve the rapid liver uptake rate of the albumin nanoplatform, thereby increasing the biological circulation half-life, thereby increasing the disease targeting rate and enhancing the usefulness as a diagnostic agent, and can reduce the impact on liver toxicity during drug delivery.

[0151]

[0152] Although some implementation forms of the present invention have been described above, the present invention is not limited to the implementation forms described above, and can be implemented by modifying and changing them within a scope that does not deviate from the gist of the present invention, and it should be understood that forms with such modifications and changes also fall within the technical spirit of the present invention.

Claims

1. Obtained by a click chemistry reaction between albumin having an azide (N3) or cyclooctyne functional group and a carrier having an azide (N3) or cyclooctyne functional group, When the above albumin is combined with an azide functional group, the transmitter is combined with a cyclooctane functional group, and when the above albumin is combined with a cyclooctane functional group, the transmitter is combined with an azide functional group. A surface-modified albumin nanoplatform, wherein at least one of the azide or cyclooctane functional groups bound to the albumin, to which a carrier is not bound, is inactivated.

2. In paragraph 1, A surface-modified albumin nanoplatform having 1 to 30 azide or cyclooctane functional groups introduced into the above albumin.

3. In paragraph 1, A surface-modified albumin nanoplatform, wherein the above inactivation is performed by bonding a masking material having a substituent selected from the group consisting of an amino group (-NH2), a carboxyl group (-COOH), and a hydroxyl group (-OH) to an azide or cyclooctane functional group bound to albumin.

4. In paragraph 3, A surface-modified albumin nanoplatform wherein the masking material is lysine or polyethylene glycol (PEG).

5. In paragraph 3, A surface-modified albumin nanoplatform, wherein the bonding of the above masking material is performed by a click chemical reaction between the masking material having an azide or cyclooctane functional group bonded and the albumin having the delivery material bonded.

6. In paragraph 5, A surface-modified albumin nanoplatform, wherein when the albumin is bonded to an azide functional group, the masking material is bonded to a cyclooctane functional group, and when the albumin is bonded to a cyclooctane functional group, the masking material is bonded to an azide functional group.

7. In paragraph 1, A surface-modified albumin nanoplatform, wherein the above-mentioned delivery substance is at least one selected from the group consisting of a sugar compound, folic acid or a derivative thereof, RGD (arginyl-glycyl-aspartic acid) or a derivative thereof, an active substance, a radioactive isotope, and a fluorescent substance.

8. In paragraph 7, A surface-modified albumin nanoplatform, wherein the above-mentioned sugar compound is at least one selected from the group consisting of a glucosyl group, a mannosyl group, and a galactosyl group.

9. In paragraph 7, The above radioactive isotope 3 H, 11 C, 18 F, 14 Cl, 32 P, 35 S, 36 Cl, 45 Ca, 51 Cr, 57 Co, 58 Co, 59 F, 64 Cu, 67 Go, 68 Go, 89 Zr, 90 Y, 99 Mo, 99 mTc, 111 In, 131 I, 125 I, 124 I, 123 I, 186 Re, 188 Re, 225 Ac, 212 Pb, 117 mSn, and 177 A surface-modified albumin nanoplatform comprising at least one selected from the group consisting of Lu.

10. In paragraph 9, The above radioactive isotope is labeled with a chelating agent, and the chelating agent is NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DFO (3-[6,17-dihyroxy-7,10,18,21-tetraoxo-27-[N-acetylhydroxylamino)-6,11,17,22-tetraazaheptaeicosane]thiourea), DTPA (diethylenetriaminepentaacetic acid), N2S2(diaminedithiol), p-SCN-Bn-NOTA (2-(4'-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid), NODAGA (1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid), p-SCN-Bn-DOTA (2-(4'-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), p-SCN-Bn-DTPA (2-(4-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid), p-SCN-Bn-DFO A surface-modified albumin nanoplatform, comprising at least one selected from the group consisting of (1-(4-Isothiocyanatophenyl)-3-[6,17-dihyroxy-7,10,18,21-tetraoxo-27-[Nacetylhydroxylamino)-6,11,17,22-tetraazaheptaeicosane]thiourea) and HYNIC (hydrazinonicotinic acid).

11. In paragraph 1, The above fluorescent substances are FNR (Ferrodoxin NADP(+) reductase), cyanine fluorescent substances, TAMRA (tetramethylrhodamine-5-maleimide), and Flamma ® A surface-modified albumin nanoplatform comprising at least one selected from the group consisting of fluorescent substances and ICG (indocyanine green). 12.(1) A step for preparing an albumin solution having an azide (N3) or cyclooctyne functional group attached; (2) a step of mixing a solution containing a carrier having a cyclooctane functional group or an azide (N3) functional group bonded to the solution prepared in the above step (1) and performing a click chemistry reaction without a copper catalyst; and (3) A step of mixing a solution containing a masking material having a cyclooctane functional group or an azide (N3) functional group with an albumin solution to which a carrier is combined, and performing a click chemistry reaction without a copper catalyst. Including, but not limited to, When the above albumin is combined with an azide functional group, the transmitter and masking agent are combined with a cyclooctane functional group, and when the above albumin is combined with a cyclooctane functional group, the transmitter and masking agent are combined with an azide functional group. Method for manufacturing surface-modified albumin nanoplatform.

13. In paragraph 12, A method for producing a surface-modified albumin nanoplatform, wherein the molar ratio of albumin and masking material in the step (3) above is 1:10 to 1:

100.

14. A composition for diagnosing or treating a disease, comprising a surface-modified albumin nanoplatform according to any one of claims 1 to 11.

15. In paragraph 14, A composition for diagnosing or treating a disease, wherein the disease is selected from acute nephritis, periodontitis, sarcopenia, pancreatic cancer, head and neck cancer, endometrial cancer, bone cancer, breast cancer, central or peripheral nervous system cancer, digestive cancer, germ cell cancer, adenocarcinoma, blood cancer, renal-urinary tract cancer, liver cancer, lung cancer, pleural cancer, prostate cancer, sarcoma, and skin cancer.

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