KR101879682B1

KR101879682B1 - Albumin-based disease targeting diagnostic or therapeutic nano-platform

Info

Publication number: KR101879682B1
Application number: KR1020160025111A
Authority: KR
Inventors: 이윤상; 강건욱; 정재민; 이동수; 정준기; 송명근; 김우형; 박지용
Original assignee: 서울대학교산학협력단
Priority date: 2015-03-11
Filing date: 2016-03-02
Publication date: 2018-07-19
Also published as: KR20160110119A

Abstract

The present invention relates to a method for producing an albumin nano platform in which albumin an azide (N ₃ ) group or a cyclooctyne group is modified, and an albumin nano platform produced thereby. The present invention also relates to a method for producing an albumin nano platform kit, an albumin nano platform in which a delivery material containing an azide (N ₃ ) group or a cyclooctyne group is bound to the albumin nano platform, Lt; RTI ID = 0.0 > albumin < / RTI > The albumin-based platform according to the present invention exists not only in the intravascular compartment but also due to its size, which prevents binding and reabsorption in the kidney, so that kidney intake can be avoided. As a result, the drug delivery and radiopharmaceuticals free from nephrotoxicity Wide application is possible. In addition, the present invention is a platform technology that facilitates effective drug delivery to target tissues and organs by minimizing deformation and being present in the intravascular compartment for a long time, thereby facilitating high intake into target tissues.

Description

[0001] The present invention relates to an albumin-based disease-targeting diagnostic or therapeutic nano-platform,

The present invention relates to a method for producing an albumin nano platform in which albumin an azide (N ₃ ) group or a cyclooctyne group is modified, and an albumin nano platform produced thereby. The present invention also relates to a kit for producing an albumin nano platform to which a delivery material containing an azide (N ₃ ) or a cyclooctyne group is bound to the albumin nano platform, a method for producing an albumin nano platform, Lt; RTI ID = 0.0 > albumin < / RTI >

Currently, nanoparticle-based methods such as albumin and other small molecule-based methods are used in practical clinical applications. Nanoparticle-based delivery and treatment is a drug delivery method using various materials such as liposomes and antibodies, and many studies have shown that inevitably high intake of the endothelial system including the liver is high.

Approaches for diagnosis and treatment using small molecules include Vintafolide as a drug polymer targeting folate receptors, and DOTATOC and DOTATATE as radioactive isotope markers targeting a somatostatin receptor in neuroendocrine tumors. Such small molecule based imaging / targeting materials are known to exhibit rapid uptake of target sites and high target / non-target uptake rates, but nephrotoxicity such as kidney toxicity occurs when the kidney is the main excretory pathway.

In particular, drug delivery, diagnosis and therapeutic applications using albumin have been applied in many fields such as medicine. Typically, in the case of albumin labeled with Tc-99m, the monomers are used to evaluate the intravascular movement and distribution of blood, and aggregates are used to assess the damage site of the lungs by blocking the capillaries of the lungs.

In addition, serum albumin, which is a folic acid-labeled radioisotope (particularly, Tc-99m) prepared for the purpose of diagnosing a surveillance lymph node containing a tumor, has been disclosed (Korean Patent No. 10-1395085). However, since albumin is transmitted through lymphatic vessels, it can not be said to overcome the problems of conventional albumin derivatives, such as ingestion of target sites and renal toxicity, and its use is merely to diagnose and visualize surveillance lymph nodes containing tumors.

In addition, a technique of forming a complex for drug delivery by combining hydrophilic albumin and hydrophobic bile acid is known (Korean Patent No. 10-1043407). However, the protein complex forms a self-aggregate in a hydrophilic environment and delivers the drug in the form of nanoparticles.

On the other hand, the conventional radioisotope labeled albumin derivatives have been hindered from accurate diagnosis because the liver is not constantly consumed in the liver. The root cause of this problem is expected to result from the modification of albumin by physico-chemical stress (high and low pH, high temperature, organic solvent, oxidizing / reducing agent) applied while introducing various functional groups on the surface of bio- have.

Under the above circumstances, the present inventors have made efforts to solve the problems of existing albumin or small molecule-based in vivo delivery nanoparticles. As a result, it has been found that by using Cu-free click chemistry, various functional groups Substances, radioactive isotopes, etc.) are introduced to minimize the denaturation of albumin, and can be used as drug delivery and radiopharmaceuticals that are present in the intravascular compartment and free from nephrotoxicity, thus completing the present invention.

It is an object of the present invention to provide a method for producing an albumin nano platform in which an albumin (N ₃ ) group or a Cyclooctyne group is modified with an albumin.

Another object of the present invention is to provide an albumin nano platform manufactured by the method for producing the albumin nano platform.

It is another object of the present invention to provide a kit for producing albumin nanoparticles in which a delivery material containing an azide (N ₃ ) or a cyclooctyne group is bound to the albumin nano platform.

Yet another object of the present invention is to provide a method for producing an albumin nano platform in which a delivery material containing an azide (N ₃ ) or a cyclooctyne group is bound to the albumin nano platform.

Another object of the present invention is to provide a delivery material-bound albumin nano platform manufactured by the method for producing the albumin nanoparticle to which the delivery material is bound.

One aspect of the present invention provides a method for producing an albumin nanoparticle in which an albumin (N ₃ ) group or a cyclooctyne group is modified with an albumin.

(A) dissolving albumin in phosphate-buffered saline (PBS) and dissolving azide (N ₃ ) -NHS or Cyclooctyne-NHS in DMSO; And (b) mixing the albumin solution prepared in the above step with a solution of azide (N ₃ ) -NHS or cyclooctyne-NHS and reacting at 20 to 37 ° C for 30 minutes to 1 hour To provide a way to manufacture albumin nano platforms.

Another aspect of the present invention provides an albumin nano platform manufactured by the method for producing the albumin nanoparticle.

Another aspect of the present invention is a method for preparing a solution comprising: (i) preparing a solution to which an azide (N ₃ ) is bonded to an N-terminal amine group of an albumin monomer or a solution to which Cyclooctyne is bound; And (ii) a solution containing a cyclodextrin-bonded transfer agent or an azide (N ₃ ) group-containing transfer agent is mixed with the solution prepared in step (i) Cu-free click chemistry. When a solution in which an azide (N ₃ ) group is bound to the albumin monomer in step (i) is used, the step (ii) (N ₃ ) -bonded transfer material in the step (ii) when a solution containing a cyclooctyne group is used in the step (i) is reacted with a solution containing a donor Wherein the albumin nanoparticle is reacted with a solution containing the albumin nanoparticle.

One embodiment of the present invention provides a method for preparing a delivery material-bound albumin nano platform, wherein the delivery material is a radioactive isotope, folate or RGD (arginyl-glycyl-aspartic acid).

Another embodiment of the present invention is a process for preparing an albumin nano platform wherein the number of azide (N ₃ ) or cyclooctyne reactors introduced into the albumin nano platform produced by the method is from 1 to 20 &Lt; / RTI >

Another embodiment of the present invention provides a method for preparing an albumin nano platform, wherein the solution of step (i) is reacted at a pH of 6.8 to a pH of 7.6 and a temperature of 20 to 37 캜 for 30 minutes to 1 hour.

Another embodiment of the present invention is a process for the preparation of 4-cyclooctyn-1-yl (Cyclooctyne)

), 3-cyclooctyn-1-yl (

), 2-cyclooctyn-1-yl (

), Monofluorinated cyclooctyne (MOFO) (

), Difluororinated cyclooctyne (DIFO) (

), Dimethoxyazacyclooctyne (DIMAC) (

), Dibenzocyclooctyne (DIBO) (

), Azadibenzocyclooctyne (ADIBO) group (

) Or Biarylazacyclooctynone (BARAC) (

). &Lt; / RTI >

Another aspect of the present invention provides a delivery material-bound albumin nano platform produced by a method of producing the albumin nanoparticle to which the delivery material is bound.

Another aspect of the present invention is a method for producing a liquid composition comprising (a) a solution containing an albumin monomer (Solution 1-1); (b) a solution containing azide (N ₃ ) or cyclooctane (first-second solution); And (c) a solution (second solution) containing a delivery material to which an azide group (N ₃ ) is bonded or a delivery material to which a cyclooctyne is bound, the kit comprising: Is characterized in that the second solution is subjected to a Cu-free click chemistry reaction with a solution obtained by reacting the 1-1 solution and the 1-2 solution, wherein the 1-2 solution is an azide N ₃ ), the second solution is a solution containing a carrier material to which a Cyclooctyne group is bonded. When the first-second solution is a solution containing Cyclooctyne, 2 solution is a solution containing a transfer material to which an azide (N ₃ ) group is bonded.

Another embodiment of the invention wherein the radioisotope ^{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 ^m Tc, ¹¹¹ In, ¹³¹ I, ¹²⁵ I, ¹²⁴ I, ¹²³ I, ¹⁸⁶ Re, ¹⁸⁸ Re and ¹⁷⁷ Lu Or more of the radioactive isotope of the present invention is a radioisotope.

Another embodiment of the present invention provides a kit for producing albumin nanoparticles, wherein the radioisotope is bound to a chelating agent.

In another embodiment of the present invention, the chelating agent is selected from the group consisting of NOTA, DOTA, DFO, DTPA, N ₂ S ₂ , p-SCN-Bn-NOTA, NODAGA, p- Bn-DTPA, p-SCN-Bn-DFO, or HYNIC.

In another embodiment of the present invention, the Cyclooctyne group is 4-cyclooctyn-1-yl (

), 3-cyclooctyn-1-yl (

), 2-cyclooctyn-1-yl (

), Monofluorinated cyclooctyne (MOFO) (

), Difluororinated cyclooctyne (DIFO) (

), Dimethoxyazacyclooctyne (DIMAC) (

), Dibenzocyclooctyne (DIBO) (

), Azadibenzocyclooctyne (ADIBO) group (

) Or biarylazacyclooctynone (BARAC) (

), A kit for manufacturing an albumin nano platform.

(A) a solution (first solution) containing an albumin monomer to which an azide (N ₃ ) is bonded or an albumin monomer to which a cyclooctane is bound; (b) a solution (second solution) containing a delivery material to which an azide (N ₃ ) group is bonded or a delivery material to which a Cyclooctyne group is bound, wherein the kit comprises 1 and a second solution are reacted by a Cu-free click chemistry without using a copper catalyst. When the first solution is a solution containing azide (N ₃ ), the second solution is a solution containing cyclooctane Wherein the first solution is a solution containing Cyclooctyne and the second solution is a solution containing a transfer material to which an azide (N ₃ ) group is bonded, A kit for manufacturing an albumin nano platform.

Hereinafter, the present invention will be described in detail.

One aspect of the invention relates to a method of making an albumin nano platform, wherein the albumin is modified with an azide (N ₃ ) group or a Cyclooctyne group. Particularly, one aspect of the present invention is a method for preparing a pharmaceutical composition comprising the steps of (a) dissolving albumin in phosphate buffer (PBS), dissolving azide (N ₃ ) -NHS or Cyclooctyne-NHS in DMSO; And (b) mixing the albumin solution prepared in the step (a) with N ₃ -NHS or Cyclooctyne-NHS solution and reacting at 20 to 37 ° C for 30 minutes to 1 hour. , And a method for manufacturing an albumin nano platform.

The term " albumin " used in the present invention is one of the proteins constituting the basic material of a cell, and refers to a protein that exists in the blood in a very large amount and is produced in the liver. The albumin has the smallest molecular weight among simple proteins existing in a natural state. Serum albumin in blood has a function of maintaining and restoring plasma volume, preventing shock due to excessive bleeding, and being used for surgery and burn treatment. It is also known to have oxygen transfer capability similar to hemoglobin. The albumin may include all albumin that can be formulated, but is preferably derived from human plasma or recombinant human serum albumin produced by genetic engineering, but is not limited thereto. The genetic information for the albumin of the present invention can be obtained from known databases such as NCBI GenBank.

In the present invention, the surface of the albumin may have 15 to 20 exposed amino groups (-NH ₂ ).

In a specific embodiment of the present invention, human serum albumin (HSA) is typically used to produce a nano platform that can be used for drug delivery, diagnosis, or treatment in vivo based on albumin having biocompatibility , Cyclooctyne or Azide (N ₃ ) group, which is one of the functional groups of click chemistry, was introduced to the surface of serum albumin as a reaction condition with minimal denaturation of HSA.

In the present invention, the azide (N ₃ ) group is a reactor composed of three nitrogen atoms and has a high reactivity. Especially, it plays a role of electron donor in the 1,3-dipolar cycloaddition reaction which is a kind of Cu- To form a triaza-5-membered ring.

In the present invention, the term Cyclooctyne refers to an 8-membered aliphatic ring containing a triple bond under ring strain. Especially, in the 1,3-dipolar cycloaddition reaction, which is a type of Cu-free click chemistry, And it is known to play a role in creating a triaza-5-membered ring. The structural characteristics of Cyclooctyne, ie the triple bond structure under ring strain, enable click chemistry without a Cu (I) catalyst. Also, in the present invention, the Cyclooctyne group is 4-cyclooctyn-1-yl (

), 3-cyclooctyn-1-yl (

), 2-cyclooctyn-1-yl (

), Monofluorinated cyclooctyne (MOFO) (

), Difluororinated cyclooctyne (DIFO) (

), Dimethoxyazacyclooctyne (DIMAC) (

), Dibenzocyclooctyne (DIBO) (

), Azadibenzocyclooctyne (ADIBO) group (

) Or biarylazacyclooctynone (BARAC) (

).

In the present invention, the pH of the phosphate buffer solution may be from pH 6.8 to pH 7.6, and it may be pH 7.0 to pH 7.4, but is not limited thereto.

Meanwhile, in the present invention, the amount of DMSO, which is a solvent required for preparing an azide (N ₃ ) -NHS solution or a cyclooctyne-NHS solution, may be 2% (v / v) or less of the total reaction solution.

In an embodiment of the present invention, when the N ₃ -NHS solution or the Cyclooctyne -NHS solution is prepared, in order to minimize the amount of DMSO relative to the total reaction volume, the total reaction solution volume 2% (v / v) or less of the total amount.

In the present invention, the mixing molar ratio of the albumin to the azide (N ₃ ) -NHS or the cyclooctyne-NHS may be 1: 1 to 1:25 in the step (b).

In addition, the number of the azide (N ₃ ) or the cyclooctane (Cyclooctyne) reactors introduced into the albumin nano platform manufactured by the method for producing the albumin nano platform of the present invention may be 1 to 20.

Since the albumin nanoparticle of the present invention can have two or more, especially about 20, reactors, it is possible to bind various binding substances to a plurality of reactors existing in the albumin nano platform of the present invention.

In an embodiment of the present invention, the number of click reaction functions on the albumin surface is controlled in order to produce an optimal nano platform which can be utilized in a click reaction and does not lose the properties inherent in albumin. The number of functional groups introduced into HSA was determined according to the reaction ratio of ADIBO-NHS or N ₃ -NHS and HSA. Specifically, the molecular weight of HSA-ADIBO or HSA-N _{3 was} measured by a MALDI-TOF mass spectrometer to quantify the number of ADIBO or azide introduced into HSA. The number of amino groups (-NH ₂ ) exposed on the surface of HSA is known to be approximately 15 to 20, and 1, 4, 8, and 14 Of ADIBO was introduced. It was confirmed that 1, 2, 6, and 13 azide were introduced through the control of the reaction ratio of N ₃ -NHS / HSA, respectively.

In another aspect, the present invention provides an albumin nano platform manufactured by a method according to the method for producing the albumin nano platform. In particular, in the present invention, the functional group bound to albumin in the albumin nano platform may be an azide (N ₃ ) group or a cyclooctyne group.

In the present invention, albumin, an azide (N ₃ ) group, a cyclooctyne group and the like are as described above.

Specifically, in the method for producing the albumin nanoparticle, in the case of the albumin nanoparticle prepared by mixing the albumin solution and the N ₃ -NHS solution in the step (b), the functional group bound to the albumin is an azide (N ₃ ) In the case of the albumin nanoparticle prepared by mixing the albumin solution and the cyclooctyne-NHS solution in step (b), the functional group bound to the albumin may be a cyclooctyne group.

In a specific example of the present invention, HSA-ADIBO and HSA-N ₃ were prepared by mixing human serum albumin solution with ADIBO-NHS solution or N ₃ -NHS solution and reacting at 37 ° C for 30 minutes.

The albumin nanoparticle of the present invention may have a cyclooctyne group or an azide group (N ₃ ) group attached to the surface of albumin constituting the albumin surface, and the number of the attached functional groups may be selected from the group consisting of albumin and Cyclooctyne- NHS or N < ₃ > -NHS may be mixed and reacted. In particular, in the present invention, the number of the azide (N ₃ ) or cyclooctylic (Cyclooctyne) reactors introduced into the albumin nano platform may be 1 to 20.

In another aspect,

(i) preparing a solution in which an azide (N ₃ ) is bonded to an N-terminal amine group of an albumin monomer or a solution to which Cyclooctyne is bound; And

(ii) a solution containing a cyclodextrin-bonded transfer agent or an azide (N ₃ ) group-containing transfer agent is mixed with the solution prepared in step (i) wherein the step (ii) comprises a step of reacting a Cyclooctyne group with a coupling agent in the step (i) wherein a solution having an azide (N ₃ ) group bound to the albumin monomer is used, (N ₃ ) -bonded transfer material is contained in the step (ii) when a solution in which a cyclooctane group is bound to the albumin monomer is used in the step (i) Wherein the albumin nanoparticles are reacted with a solution of a microcrystalline nanoparticle.

Since the azide (N ₃ ) group used as a reactor in the present invention is an electron donor and the Cyclooctyne group is an electron acceptor, in step (i), an azide (N ₃ ) group-containing solution is used, the step (ii) is performed in the step (i) by reacting a solution containing a Cyclooctyne group-conjugated transferring material, and a step of reacting the albumin monomer with a cyclooctyne group When using the combined solution, it is effective to react with the solution containing the transfer agent to which the azide (N ₃ ) group is bonded in the step (ii).

In the present invention, albumin, an azide (N ₃ ) group, a cyclooctyl group, and the like are as described above.

In the present invention, the number of the azide (N ₃ ) or the cyclooctane (Cyclooctyne) reactors introduced into the albumin nano platform manufactured by the above method may be 1 to 20.

In the present invention, the solution of step (i) may be prepared by reacting the solution at a pH of 6.8 to a pH of 7.6 and a temperature of 20 to 37 캜 for 30 minutes to 1 hour.

In another embodiment of the present invention, the solution in which the azide (N ₃ ) is bound to the N-terminal amine group of the albumin of the step (i) or the solution in which the cyclooctane is bound is (a) albumin Dissolving azide (N ₃ ) -NHS or Cyclooctyne-NHS in DMSO dissolved in phosphate buffered saline (PBS), and (b) incubating the albumin solution prepared in step (a) with N ₃ -NHS Or Cyclooctyne-NHS solution, and reacting at 20 to 37 占 폚 for 30 minutes to 1 hour to prepare an albumin nanoparticle.

Another embodiment of the present invention provides a method of producing a transfer material-bound albumin nanoparticle comprising separating the albumin nanoparticle bound to the transfer material prepared in step (ii).

Since the albumin nanoparticle of the present invention can have two or more, especially about 20, reactors, it is possible to bind various binding substances to a plurality of reactors existing in the albumin nano platform of the present invention. The binding substance that can be bound to the albumin nano platform of the present invention may be a single substance or a combination of various binding substances.

On the other hand, when the substance to be coupled to the albumin nano platform is a combination of two or more substances, a combination of two or more substances may be bound to the albumin nano platform at the same time, or each substance may be sequentially bound, .

In the present invention, the transfer substance refers to a substance that binds to the albumin nano platform and is delivered in vivo, and may be selected from the group consisting of a radioisotope, a fluorescent substance, a target molecule, an active substance, and a combination thereof. Specifically, the radioactive isotope of the present invention may be a radioactive isotope, a fluorescent substance, a target molecule, a single substance of the active substance, or a radioisotope and a fluorescent substance; Radioactive isotopes and target molecules; Radioactive isotopes and active substances; Fluorescent materials and target molecules; Fluorescent materials and active materials; Target molecules and active substances; Radioisotopes, fluorescent substances and target molecules; Radioactive isotopes, target molecules and active substances; Radioactive isotopes, fluorescent substances and active substances; A fluorescent molecule, a target molecule, an active substance or a radioactive isotope, a fluorescent substance, a target molecule and an active substance.

In the present invention, the term " isotope " refers to an element having the same atomic number but different atomic mass, and isotopes having radioactivity are referred to as " radioactive isotopes ", and gamma rays or other subatomic particles are emitted to perform radiative attenuation Is an important marker for diagnosing diseases in general. Radioisotopes that can be used in the present invention as markers and can be used without long as is known in the art ^{restrictions, 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, 99m Tc, 111 In, 131 I, 125 I, 124 I, 123 I, 186 Re, 188 Re And ¹⁷⁷ Lu. For example, the radioactive isotope of the present invention may be a diagnostic radioactive isotope such as ¹¹ C, ¹⁸ F, ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁹ Zr, ^99m Tc, ¹¹¹ In and ¹²³ I, May be elements such as ⁹⁰ Y, ¹⁷⁷ Lu, and ¹⁸⁸ Re, and in particular, it may be ⁶⁴ Cu or ¹⁷⁷ Lu, but is not limited thereto.

In the present invention, a chelating agent may be used, which serves to link a radioisotope to albumin. Examples of the chelating agent include 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-dihydroxy-7,10,18,21-tetraoxo-27- [N- acetylhydroxylamino ) -6,11,17,22-tetraazaheptaeicosane] thiourea), DTPA (diethylenetriaminepentaacetic acid), N ₂ S ₂ (diaminedithiol), p-SCN-Bn-NOTA (2- (4'-isothiocyanatobenzyl) 7-triazacyclononane-1,4,7-triacetic acid), NODAGA (1,4,7-triazacyclononane, 1-glutaric acid-4,7-acetic acid) 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 (1- (4-Isothiocyanatophenyl) -3- [6,17- 21-tetraoxo-27- [N-acetylhydroxylamino] -6,11,17,22-tetraazaheptaeicos ane] thiourea, or HYNIC (hydrazinonicotinic acid).

The term " fluorescent substance " in the present invention means a substance that emits a specific wavelength of visible light with respect to a specific wavelength. In the present invention, the fluorescent substance includes all fluorescent substances that can be used to confirm the position of the nano platform. Specifically, fluorescent materials that can be used as a marker in the present invention can be used without limitation as long as they are known in the art. Rhodamine systems including rhodamine, TAMRA and the like; Fluorescein, including fluorescein, fluorescein isothiocyanate (FITC) and fluorecein amidite (FAM); Bodipy (boron-dipyrromethene); Alexa fluor system; And cyanine including Cy3, Cy5, Cy7, indocyanine green, etc. However, the present invention is not limited thereto.

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 induces the specific environment of the albumin nano platform of the present invention . The target molecule of the present invention can induce the albumin nano platform of the present invention to a specific environment, determine the presence or absence of the specific environment, the location of the specific environment, and the degree of the specific environment. The specific environment in vivo targeted by the target molecule in the present invention may be a tissue-specific specific environment or a disease-specific specific environment. That is, the target molecule may be a substance that targets a tissue-specific substance or a substance that is disease-specific. A substance that exists in a tissue-specific state may be a protein or RNA that is tissue-specific, a compound that accumulates or is produced, and the substance that exists in a disease-specific state is a protein, RNA, And so on. In particular, in the present invention, the target molecule may be folate or a derivative thereof or an arginyl-glycyl-aspartic acid (RGD) or derivative thereof which targets a folate receptor present in a large number of cancer tissues.

&Quot; Folic acid " of the present invention is a kind of vitamin, which is also called vitamin B9 or vitamin M. Folic acid binds specifically to folate receptors. A "folate receptor" is a tumor-associated glycosyl phosphatidylinositol anchor protein that allows a conjugated folic acid or a folate-bound material to be taken up via receptor-mediated endocytosis.

The "RGD" of the present invention is a tripeptide consisting of three amino acids, namely, L-arginine, glycine, and L-aspartic acid. Found. Because they have a strong affinity for the integrin receptor, which is mainly expressed in the endothelial cell layer of tumor vasculature, it is widely used as a universal means of research such as drug targeting treatment and simple reagent.

To bind to albumin to transfer material in the present invention, CONH, NHCO, NHCONH, NHCSNH, OCONH, NHOCO, S, O, CO, (C = O) O, O (C = O), OCH 2 CH 2, CH 2 A linker consisting of one or more selected from the group consisting of CH ₂ O, NH (CNH), (CNH) NH, NN and NH can be used.

The tissue in the present invention may be a tissue having a tissue specific property and may be a tissue having specific characteristics such as brain, eye, nerve, bone, lung, liver, stomach, heart, blood vessel, muscle, large intestine, small intestine, duodenum, Pancreas, urethra, skin, ovary, respiratory tract, auditory organ, and the like.

In the present invention, the disease may be a disease in which a disease-related in vivo part has disease-specific properties, and may be selected from the group consisting of cardiovascular diseases including cancer, atherosclerosis, neovascularization diseases, and combinations thereof , But is not limited thereto. The cancer may be a primary cancer or a metastatic cancer.

The term " active substance " of the present invention may be a natural product, a vitamin, an active agent, a biologic, a compound, an amino acid, a protein, a peptide, a nucleotide, a nucleic acid, a polypeptide, a polynucleotide and analogues, An antidiarrheal agent, an antibiotic, an anticoagulant, an antidepressant, an antidiabetic agent, an anti-epileptic agent, an antihistamine agent, an anti-inflammatory agent, an anti-inflammatory agent, An antihypertensive agent, an antihypertensive agent, an antimuscarinic agent, an anti-mycobacterial agent, an immunosuppressive agent, an antithyroid agent, an antiviral agent, an anti-anxiolytic agent, a sedative agent, an astringent agent, an alpha-adrenergic receptor blocker, a beta-adrenergic receptor blocker, Contrast media, cough suppressants, diagnostic reagents, diagnostic imaging agents, diuretics, dopamine agonists, hemostats, immunizations And may be selected from the group consisting of an agent, a lipid regulator, a muscle relaxant, a parasympathetic stimulant, an antiallergic agent, an appetite suppressant, a sympathetic stimulant, a thyroid agent, a vasodilator, and combinations thereof.

In a specific embodiment of the present invention, an albumin nano platform in which a radioactive isotope, Cu-64, representative of the albumin nano platform of the present invention, and a folate targeting a folate receptor existing in a cancer tissue are combined with a transfer material is manufactured Respectively. Specifically, the radioactive isotope labeled with NOTA-N ₃ by using Cu-64 was added to HSA-ADIBO or HSA-N ₃ and HSA-folate to obtain Cu-64-NOTA-N ₃ To prepare an HSA-Folate derivative.

In another specific embodiment of the present invention, a cyclic RGD (arginyl-glycyl-aspartic acid) targeting the integrin? V? 3 expressed in cancer tissue and Cu-64, a typical radioisotope in the albumin nano platform of the present invention, Nano platform was manufactured. Specifically, after the labeling radioisotope the NOTA-N ₃ using the Cu-64, this HSA-ADIBO or HSA-N _3, and the result of the addition in response to the HSA-RGD, Cu-64- NOTA-N 3 HSA-RGD < / RTI >

In another aspect, the present invention provides a delivery material-bound albumin nano platform manufactured by a method of producing an albumin nanoparticle to which the delivery material is bound.

In the present invention, albumin, a transmitter, and the like are as described above.

In yet another aspect,

(a) a solution containing an albumin monomer (Solution 1-1);

(b) a solution containing azide (N ₃ ) or Cyclooctyne (Solution 1-2); And

(c) a solution (second solution) containing a delivery material to which an azide group (N ₃ ) is bonded or a delivery material to which a cyclooctyne is bound, the kit comprising: Characterized in that the second solution is subjected to Cu-free click chemistry reaction with a solution obtained by reacting the 1-1 solution and the 1-2 solution, wherein the 1-2 solution is an azide (N ₃ ), the second solution is a solution containing a carrier material to which a Cyclooctyne group is bound, and when the first-second solution is a solution containing Cyclooctyne, Wherein the solution is a solution containing a carrier material to which an azide (N ₃ ) group is bonded.

In the present invention, albumin, an azide (N ₃ ) group, a cyclooctyne group, a transporter and the like are as described above.

DOTA, DFO, DTPA, N ₂ S ₂ , p-SCN-Bn-NOTA, NODAGA, p-SCN-Bn -DOTA, TETA, p-SCN-Bn-DTPA, p-SCN-Bn-DFO or HYNIC.

In yet another aspect,

(a) a solution (first solution) containing an albumin monomer bonded with azide (N ₃ ) or an albumin monomer conjugated with cyclooctyne;

(b) a solution (second solution) containing a delivery material to which an azide (N ₃ ) group is bonded or a delivery material to which a Cyclooctyne group is bound, wherein the kit comprises 1 and a second solution are reacted by a Cu-free click chemistry without using a copper catalyst. When the first solution is a solution containing azide (N ₃ ), the second solution is a solution containing cyclooctane Wherein the first solution is a solution containing Cyclooctyne and the second solution is a solution containing a transfer material to which an azide (N ₃ ) group is bonded, A kit for manufacturing an albumin nano platform.

The albumin-based platform according to the present invention exists not only in the intravascular compartment but also due to its size, which prevents binding and reabsorption in the kidney, so that kidney intake can be avoided. As a result, the drug delivery and radiopharmaceuticals free from nephrotoxicity Wide application is possible. In addition, the present invention is a platform technology that facilitates effective drug delivery to target tissues and organs by minimizing deformation and being present in the intravascular compartment for a long time, thereby facilitating high intake into target tissues.

FIG. 1 discloses a process for producing an albumin nano platform. Specifically, a method for producing HSA-ADIBO or HSA-N ₃ by reacting an amino group present on the surface of albumin with ADIBO-NHS or N ₃ -NHS is disclosed .
FIG. 2 shows the results of measurement of the molecular weight of HSA-ADIBO by MALDI-TOF (Matrix-assisted laser desorption / ionization-time of flight) mass spectrometer according to the reaction ratio of albumin to ADIBO-NHS.
FIG. 3 is a graph showing the results of measurement of the peak movement with increasing molecular weight by a MALDI-TOF mass spectrometer in reactions of the first type to the fourth type in which the reaction ratios of albumin and ADIBO-NHS were different.
FIG. 4 shows the results of measurement of the molecular weight of HSA-N ₃ by the MALDI-TOF mass spectrometer according to the reaction ratio of albumin to N ₃ -NHS.
5 is treated with labeled albumin nano platform that combines a NOTA-N ₃ labeled with a radioactive isotope (Ga-68, Cu-64 and Lu-177) in the HSA-ADIBO through the tail vein of normal animals (mice) This is a video image that measures radioactivity in vivo over time.
FIG. 6 is a graph showing the results of the administration of NOTA-ADIBO labeled isotope labeled albumin nano platform labeled with radioactive isotopes (Ga-68, Cu-64 and Lu-177) to HSA-N ₃ via the tail vein of normal animals This is a video image that measures radioactivity in vivo over time.
FIG. 7 is a screen image of a KB cell line when FNR648-Albumin-Folate conjugated with Folate, FNR648-Albumin-Folate, and FNR648-Albumin-Folate are blocked.
FIG. 8 is an image of the in vivo radioactivity measurement of the axonal tumor mouse model after administration of Cu-64 radioisotope-labeled HSA-Folate via the tail vein.
FIG. 9 is a picture of the in vivo radioactivity measurement of a peritoneal cancer metastasis model mouse after administration of Cu-64 radioisotope-labeled HSA-folate via the tail vein.
FIG. 10 shows the results of the in vivo analysis of Cu-64-Albumin, Cu-64-Albumin-Folate, Cu-64-Albumin-Folate, This is the target image of the experiment.
FIG. 11A is an image obtained by confocal laser measurement of the degree of binding of cRGDyK-albumin in the cell membrane and in the cell in the reaction A and the reaction B. FIG.
FIG. 11B is an image screen of the reaction of A and B in which the degree of binding of albumin to the cell membrane and the inside of the cell is measured by confocal laser.
FIG. 12A is a photograph of a PET image of a cervical intravenous injection of Cu-64-labeled cRGDyK-albumin in a 6-week-old mouse tumor model.
FIG. 12B is a photograph of a PET image obtained after administration of a cervical intravenous injection of Cu-64 labeled cRGDyK-albumin in a 6-week-old mouse tumor model.
Figure 13 is a graph of the% ID / g value over time in reactions A and B;
Fig. 14 is a conceptual diagram of the albumin nano platform of the present invention, particularly a conceptual diagram illustrating examples and advantages of components that can be combined with the structural diagram of the albumin nano platform of the present invention.

Hereinafter, the present invention will be described in more detail with reference to examples. It is to be noted, however, that these examples are illustrative of some experimental methods and compositions for illustrative purposes of the present invention, and the scope of the present invention is not limited to these examples.

Example 1: Preparation of albumin nano platform

The present inventors have attempted to produce a nano platform that can be used for drug delivery or diagnosis or treatment in vivo based on albumin having biocompatibility. Azadibenzocyclooctyne (ADIBO) or azide (N ₃ ) group, which is one of the functions of click chemistry on the surface of human serum albumin, was denatured with HSA (human serum albumin, HSA) To minimize reaction conditions.

Specifically, the albumin nano platform was prepared through the following procedure.

1-1. Preparation of albumin and introduction of ADIBO functional group

(1) Preparation of albumin solution and DMSO solution prepared by dissolving ADIBO-PEG4-NHS

First, human serum albumin (HSA) was purchased from Green Cross Medical Foundation, and 100 mL of 20% human serum albumin (Green Cross-Albumin Co., Ltd.), a specialty drug sold in liquid form as a 20% / 50 mu l to prepare albumin solution.

ADIBO-NHS was purchased from FutureCAM (FC-6123) and dissolved in DMSO at a concentration of 2 mg / 50 μl to prepare ADIBO-NHS solution.

However, when preparing the ADIBO-NHS solution, DMSO was prepared so as to contain 2% (v / v) or less of the total volume of the reaction solution so that the amount of DMSO was minimized relative to the total reaction volume.

Next, 5 mg / 50 μl of HSA solution was added to Ependorf (EP) tube, and PBS (pH 7.4 or more) was added to make 500 μl (A sample). In addition, 2 mg / 50 μl of ADIBO-NHS solution dissolved in DMSO was added to a separate EP tube in accordance with the molar ratio and made into 500 μl using PBS as described above (B sample). When preparing the B sample, the solution is poured into the EP tube by using the wall of the tube, and vortexing is performed immediately to disperse it. The A sample and the B sample were mixed and reacted at 37 DEG C for 30 minutes using a Tip type thermostat.

(2) Albumin surface functional group number regulation

The purpose of this study was to control the number of click - reaction functional groups on the albumin surface in order to produce an optimal nano platform which can be utilized in the click reaction and does not lose the properties inherent in albumin.

For this purpose, the number of ADIBO functional groups introduced into HSA was determined according to the reaction ratio of ADIBO-NHS to HSA. Specifically, the molecular weight of HSA-ADIBO was measured by MASDI-TOF (Matrix-assisted laser desorption / ionization-time of flight) mass spectrometer and the number of ADIBO introduced into HSA was quantified. The results are shown in Table 1 below (Fig. 2).

Mass (peak)
Mass value-HSA mass
Number of ADIBO adoption
Reaction molar ratio
(ADIBO-NHS / HSA)
% conjugation
HSA
66692
Reaction 1
(RXN # 1)
66982
290
One
1.12
89.28571
Reaction 2
(RXN # 2)
67894
1202
4.14483
5.56
74.54676
Reaction 3
(RXN # 3)
69059
2367
8.16207
11.24
72.65302
Reaction 4
(RXN # 4)
70748
4056
13.9862
22.47
62.24388

The number of amino groups (-NH ₂ ) exposed on the surface of HSA is known to be approximately 15 to 20, and 1, 4, 8, and 14 Of ADIBO was introduced.

In order to evaluate the physical properties, four types of reactions were newly performed by adjusting the reaction ratio of ADIBO-NHS / Albumin to determine the degree of functionalization (DOF), the size of the albumin nano platform and the zeta potential potential. Specifically, the degree of functionalization through the average molecular weight was confirmed using MALDI-TOF, and the size and zeta potential of the albumin nano platform were confirmed through dynamic laser scattering (DLS) equipment.

MALDI-TOF results for four types of reactions were confirmed, and peaks were observed to move with increasing molecular weight (FIG. 3). The DOF according to the reaction rate is shown in Table 2 below.

Mw (molecular weight)
Mw-AL
(Molecular weight - albumin molecular weight)
DOF (degree of functionalization)
Albumin
66419.5
0
0
Type 1
67470.9
1051.5
2.347098
Type 2
68132.1
1712.7
3.822991
Type 3
69324.2
2904.8
6.483929
Type 4
71860.8
5441.4
12.14598

[ ADIBO - Albumin Molecular weight measurement Zeta potential Measure]

Using a dynamic laser scattering system, the change in zeta potential according to the DOF was checked based on albumin without any treatment. The results are shown in Table 3 below.

Albumin
DOF
Zeta Difference Value
size
Type 1
2.347098
-0.6
No change
Type 2
3.822991
3.7
No change
Type 3
6.483929
5.5
No change
Type 4
12.14598
2.9
Aggregation (> 50 nm)

It was confirmed that the surface charge changes according to the DOF, and the magnitude of the change is proportional to the DOF value. At this time, it was observed that the size of the fourth type reaction exceeded 50 nm, which is due to the aggregation of the hydrophobic ADIBO group, from which the surface charge of the fourth type reaction does not reflect the DOF.

1-2. Albumin preparation and Azide Function machine Introduction

(1) Preparation of albumin solution and DMSO solution prepared by dissolving N ₃ -NHS

First, human serum albumin (HSA) was purchased from the Green Cross Medical Foundation using a reagent (ditto) sold in 20% liquid form and dissolved in PBS at a concentration of 5 mg / 50 μl to prepare albumin solution.

In addition, N ₃ -NHS was purchased from Futurechem (FC-6048) and dissolved in DMSO at a concentration of 2 mg / 50 μl to prepare ADIBO-NHS solution.

However, when the N ₃ -NHS solution was prepared, the amount of DMSO was set to be 2% (v / v) or less of the total volume of the reaction solution so that the amount of DMSO was minimized relative to the total reaction volume.

Next, 5 mg / 50 μl of HSA solution was added to Ependorf (EP) tube, and PBS (pH 7.4 or more) was added to make 500 μl (A sample). In addition, 2 mg / 50 μl of N ₃ -NHS solution dissolved in DMSO was added to a separate EP tube in accordance with the molar ratio and made to 500 μl using PBS as described above (C sample). When preparing the C sample, the solution is poured into the EP tube using the wall of the tube and vortexed immediately to disperse it rapidly. The A sample and the C sample were mixed and reacted at 37 DEG C for 30 minutes using a Tip type thermostat.

(2) Albumin surface functional group number regulation

For this purpose, the number of azide functional groups introduced into HSA was determined according to the reaction ratio of N ₃ -NHS to HSA. Specifically, the molecular weight of HSA-N _{3 was} measured by a MALDI-TOF mass spectrometer to quantify the number of azides introduced into HSA. The results are shown in Table 4 below (Fig. 4).

Mass (peak)
Mass value-HSA mass
Number of azide introduced
Reaction molar ratio
(N ₃ -NHS / HSA)
% conjugation
HSA
66742
Reaction 1
(RXN # 1)
66885
143
One
1.12
89.29
Reaction 2
(RXN # 2)
67149
407
2.85
5.56
51.19
Reaction 3
(RXN # 3)
67607
865
6.05
11.24
53.82
Reaction 4
(RXN # 4)
68569
1827
12.78
22.47
56.86

The number of amino groups (-NH ₂₎ exposed to the HSA surface is known about 15 to 20 pieces, the administration to the reaction -NHS N ₃ / through the reaction unregulated of HSA 1 gae respectively, 2, 6, 13 Of azide was introduced.

Example 2: based on albumin nano platform Disease target substance And active substance binding

2-1. Albumin surface ADIBO - Functional Utilized Folate receptor Target material binding

The albumin nanoparticle was prepared by binding the delivery material using the ADIBO-functional group of the albumin platform prepared in Example 1 above. In this experiment, a folate derivative targeting the folate receptor, which is known to be overexpressed in cells of various carcinoma tissues, was conjugated as a transmitter. In this experiment, folate derivatives were synthesized by introducing an azide - functional group into the amino acid side chain of N - terminal.

First, a small amount of sodium tartrate dibasic dihydrate was added to the folate-N ₃ solution to improve the low reactivity due to the low water solubility of Folate-N ₃ . HSA-ADIBO or HSA-N ₃ (40 nmol) was dissolved in PBS (300 μl) and mixed with folate-N ₃ or folate-ADIBO (10 nmol) and reacted at 37 ° C for 1 hour.

After the reaction, only the folate-N ₃ -bonded HSA-ADIBO (HSA-Folate) derivative was isolated through PD-10 (desalting) column.

2-2. Albumin surface ADIBO - Functional Utilized RGD Target material binding

The albumin nanoparticle was prepared by binding the delivery material using the ADIBO-functional group of the albumin platform prepared in Example 1 above. In the present experiment, cyclic RGDyK derivatives were bound to confirm integrin αvβ3 targeting ability as a delivery material. In this experiment, cyclic RGDyK derivatives were synthesized by introducing ADIBO-functional group into cyclic RGDyK, which was used to confirm the performance of integrin αvβ3 target.

HSA-ADIBO (40 nmol) was dissolved in PBS (300 μl), mixed with cyclic RGDyK-N _3, and reacted at 4 ° C for 24 hours.

After the reaction, only cyclic RGDyK-N ₃ -bonded HSA-ADIBO (HSA-cyclic RGDyK) derivatives were isolated through a PD-10 (desalting) column.

The HSA-ADIBO the embodiment 1-1 of the reaction in Table 1 2 (RXN # 2), the reaction 3 (RXN # 3), the reaction 4 was used (RXN # 4), cyclic RGDyK -N 3 Details of the coupling reaction The changes in molecular weight according to the reaction are shown in Table 5 below.

HSA-ADIBO
Peak (Mw)
(-each)
DOF
Rxn # 2
67688.79
1388.79
2.933155
Rxn # 3
69365.25
3065.25
6.473874
Rxn # 4
71848.11
5548.11
11.71773
HSA-cRGDYK
Peak (Mw)
(-each)
DOF
Rxn # 2
69109.8
1421.01
1.591829
Rxn # 3
73019.2
3653.95
4.09319
Rxn # 4
77700.41
5852.3
6.555803

Example 3: Based on albumin nano platform Radioisotope sign

3-1. ADIBO - or N ₃ - chelating agents Radioisotope sign

In this example, NOTA-N ₃ was labeled with radioactive isotopes using Cu-64 and bound to HSA-ADIBO, HSA-folate and HSA-RGD.

Specifically, Cu-64 HCl solution was dried by blowing high purity nitrogen (99.999%) while vaporizing HCl of Cu-64 radioisotope solution present in HCl solution to facilitate labeling with Cu-64 radioactive isotope .

After adjusting the pH to 5 with 200 μl of 1 M sodium acetate at pH 5.3, 10 nmol of NOTA-N ₃ solution (less than 10 μl) was added and reacted at 37 ° C for 20 minutes. Afterwards, it was confirmed that the radioactive isotopes were all cleaved to NOTA-N ₃ by thin layer chromatography in 0.1 M citrate and 0.1 M sodium carbonate solution.

3-2. With albumin ADIBO - or N ₃ - Radioactive isotope Combination

(1) HSA-ADIBO and radioactive isotope labeled NOTA-N ₃

1 nmol (20 μl) of labeled Cu-64 labeled NOTA-N ₃ was added to 300 μl of HSA-Albumin, HSA-Folate or HSA-RGD and reacted at 37 ° C. for 60 minutes. A PD-10 (desalting) column To obtain HSA-Folate and HSA-RGD derivatives coupled with Cu-64-NOTA-N ₃ .

(2) HSA-N ₃ and radioactive isotope labeled NOTA-ADIBO

If addition method of combining folate-N _3, and radioisotope-labeled NOTA is _N-3, using the HSA-ADIBO using HSA-N _3, N _3-folate instead of folate-ADIBO and RGD-ADIBO, and NOTA- With ADIBO, you can proceed as follows under the same conditions.

1 nmol (20 μl) of labeled Cu-64 labeled NOTA-ADIBO was added to 300 μl of HSA-N ₃ , reacted at 37 ° C for 60 minutes, and then transferred to a Cu-64 NOTA- ADIBO-coupled HSA-Folate and HSA-RGD derivatives were isolated and obtained.

3-3. With albumin Fluorescent Combination

You can make a FNR648 _N-3 of fluorescence is combined FNR648 HSA-conjugate using the HSA-ADIBO. The 100 nmol / Prepare 10 ㎕ of FNR648-N _3, taken by volume (㎕) that corresponds to the mole number of the same amount of the fluorescent substance of the HSA-ADIBO thereto by HSA-ADIBO added to a solution of 60 min at 37 ℃ was then obtained by separating only PD-10 (desalting) is _FNR648-3 N-linked HSA-FNR648 complexes through a column. In the same manner, HSA-folate derivatives and HSA-RGD derivatives were used in place of HSA-ADIBO to produce complexes FNR648-Albumin-folate and FNR648-Albumin-RGD, in which fluorescence and target substance were simultaneously bound.

Example 4: Normal animal and animal model imaging experiments of albumin nano platform

4-1. Albumin imaging in normal animals

(1) Isotope labeling Using HSA-ADIBO Normal animal radioactivity measurement

The isotope labeled HSA-ADIBO labeled with three different isotopes (Ga-68, Cu-64 or Lu-177) in HSA-ADIBO (RXN1, RXN2, RXN3 and RXN4) (Normal group). We observed changes in the in vivo distribution by time-of-flight (PET) (positron emission tomography) or SPECT (single-photon emission computed tomography) equipment.

As a result, we can confirm that the radioactivity is clearly measured in the blood pool immediately after the administration, and the stronger the radioactivity signal is measured not only in the liver tissue but also in the lymph node as the isotope label HSA-ADIBO having a large number of ADIBO functional groups bound to the albumin surface (Fig. 5).

(2) Measurement of radioactivity of normal animals using isotope labeled HSA-N ₃

(HSA-N) labeled with three different isotopes (Ga-68, Cu-64 or Lu-177) on HSA-N ₃ (RXN1, RXN2, RXN3 and RXN4) ₃ were administered to the mice (normal group) by the tail vein and the changes in the in vivo distribution by time of PET or SPECT were observed.

As a result, it was confirmed that the radioactivity was clearly measured in the blood pool immediately after administration, and it was confirmed that strong radioactivity signals were measured in the liver tissues as the isotope label HSA-N ₃ having a large number of azide functional groups attached to the albumin surface (Fig. 6)

4-2. Tumor / new vessel target Albumin imaging

(1) Target image evaluation of folate receptor expression tumor using folate-albumin

To confirm whether the albumin associated with the folate of the present invention is targeted to tumor tissues, a tumor animal model was used.

In addition to the animal experiments, specific target images of the cell line using the FNR648-Albumin-Folate obtained in Example 3 were confirmed for confirming the target function in KB cells used for the animal model (Fig. 7).

As shown in FIG. 7, FNR648-Albumin-Folate conjugated with Folate-conjugated FNR648-Albumin-Folate was used as a blocking agent (FNR648-Albumin-Folate 100 times more than that of the control). On the other hand, in the cell line treated with FNR648-Albumin-Folate, a clear target image was confirmed.

Animal images were obtained as follows. First, HSA-Folate labeled with Cu-64 isotope was administered via the tail vein of axillary tumor mouse model, and the in vivo activity of albumin was measured by PET or SPECT equipment. Specifically, radioactivity in vivo was measured according to time (0 h, 4 h, 18 h, 24 h, and 48 h) after administration of Cu-64 labeled HSA-Folate.

As a result, it was confirmed that radioactivity was clearly measured in the blood pool from immediately after administration to 24 hours, and strong radioactivity accumulation was observed in tumor tissue from 4 hours to 48 hours, and HSA-folate according to the present invention was found to be specific for tumor tissue (Fig. 8).

Next, HSA-folate labeled with Cu-64 isotope was administered through the tail vein of the peritoneal cancer metastatic mouse model, and the in vivo activity of the albumin was measured using PET or SPECT equipment. Specifically, the radioactivity in vivo was measured according to time (0 h, 4 h, 24 h, and 48 h) after administration of Cu-64 labeled HSA-Folate.

As a result, it was confirmed that radioactivity was clearly measured in the blood pool from immediately after administration to 24 hours, and diffuse and nodular ingestion was clearly observed in the abdominal cavity from 4 hours to 48 hours, and HSA-folate Tumor-specific accumulation (Fig. 9).

In order to confirm the specificity of the target, Cu-64-Albumin-Folate in the case of using Cu-64-Albumin-Folate in which folate was bound and folate- (100-fold higher than that of Cu-64-Albumin-Folate treated with folic acid), and it was confirmed to be consistent with the results of the cell line data obtained in FIG. 7 (FIG. 10).

(2) Target image evaluation of integrin αvβ3-expressing cell line tumors using cyclic RGDyK-albumin

To confirm whether cRGDyK-albumin binds at the cell level, the cells were treated with fluorescently labeled albumin and cRGDyK-albumin and their distribution was confirmed using a confocal microscope (Figs. 11A and 11B).

Reactions A and B in this experiment refer to Reaction 2 (RXN # 2) and Reaction 3 (RXN # 3) of Example 2-2, respectively.

When cRGDyK-albumin was compared with Reaction A and Reaction B, it was confirmed that Reaction B having more cRGDyK was present in the cell membrane and the cell interior. In addition, in order to confirm that cRGDyK-albumin binds to cells by cRGDyK, a group treated with cRGDyK-excess was treated before treatment with fluorescently labeled cRGDyK-albumin, together with the group treated with cRGDyK-albumin labeled with fluorescence And the distribution in the cells was confirmed. As a result, it was confirmed that cRGDyK-albumin distributed in cells was reduced in the group treated with an excessive amount of cRGDyK. In addition, it was confirmed that the cRGDyK-albumin present in the cells was significantly reduced in the case of Reaction B compared to Reaction A when the cells were over-pretreated with cRGDyK (Fig. 11A).

In order to confirm cell binding by albumin, a group treated with the same treatment of fluorescently labeled albumin also proceeded. As a result, it was confirmed that there was a distribution around the cell by albumin, which was much reduced when the albumin was pretreated in an excessive amount. In addition, the binding by albumin did not show a significant difference in reaction A and reaction B. In the case of Reaction A, there was no significant difference in the degree of cell binding between albumin and cRGDyK-albumin. In Reaction B, cRGDyK-albumin was much more abundant in the cell membrane (Fig. 11 (b)).

When the above results are summarized, it can be confirmed that Reaction B, in which cRGDyK is more bound in the case of cRGDyK-albumin, is more bound to the cells by cRGDyK. It was found that cRGDyK-albumin was bound to cells by cRGDyK.

In order to examine the in vivo distribution pattern and integrin αvβ3 targeting ability of isotope-labeled cRGDyK-albumin, SK-OV3 cell line expressing integrin αvβ3 on the right leg of a 6-week old mouse was planted at 4 × 10 6 cells / Respectively. After 3 to 4 weeks, the tumors were injected through the tail vein into the tumor model mice with albumin labeled with Cu-64 and cRGDyK-albumin at the time of 5-10 mm in diameter. After 10 minutes, 4 hours, 24 hours and 48 hours, PET images were obtained by time (Figs. 12A and 12B).

In the case of reaction A, the distribution of Cu-64 labeled albumin and cRGDyK-albumin in the tumor was almost similar, but in the case of reaction B, cRGDyK-albumin accumulated more in the tumor, And the highest at 4 hours.

For more accurate analysis, the% ID / g value was calculated to determine the extent of accumulation of Cu-64-HSA-RGDyK injected into the tumor site by setting the region of interest in the tumor in the image. As a result, the highest value was found in cRGDyK-albumin of Reaction B, which was highest at 5.37 ± 1.09% ID / g at 4 hours. Thus, it was confirmed that cRGDyK can target and image tumors when cRGDyK is abundant (FIG. 13).

(3) Target image evaluation of SPARC-overexpressing cell line tumors using ADIBO-albumin

As can be seen from the above results, the albumin nano platform of the present invention can transmit various substances such as radioactive isotope and disease target molecule at one time, and it can be confirmed that the activity and persistence thereof are excellent. Specifically, the albumin nano platform of the present invention increases the staying time of a substance, reduces ingestion to other organs other than the target organ (less new discharge), can transmit various substances in combination, There is an advantage that it can be used as a disease target platform which can be used for treatment (Fig. 14).

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims

A solution containing an albumin monomer (Solution 1-1);
A solution containing azide (N ₃ ) or Cyclooctyne (first-second solution); And
1. A kit for producing an albumin nano platform, comprising a solution (second solution) containing a transfer substance to which an azide group (N ₃ ) is bonded or a transfer substance to which a cyclooctane group is bonded,
The kit is characterized in that the second solution is subjected to a Cu-free click chemistry reaction at pH 6.8 to 7.6 in a solution obtained by reacting the 1-1 solution and 1-2 solution,
If the first solution is a solution containing azide (N ₃ ), the second solution is a solution containing a transfer material to which a Cyclooctyne group is bonded, and the first solution is a solution containing a cyclooctane (Cyclooctyne), the second solution is a solution containing a transfer material to which an azide (N ₃ ) group is bonded,
The reaction molar ratio of the albumin monomer of the solution 1-1 to the azide (N ₃ ) or the cyclooctane (Cyclooctyne) of the solution 1-2 is 1: 1 to 1:12,
Wherein the transmitter is a radioisotope bound to a chelating agent at a pH of 5.

delete

The method of claim 1, wherein 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 One or more elements selected from the group consisting of Ga, ⁶⁸ Ga, ⁸⁹ Zr, ⁹⁰ Y, ⁹⁹ Mo, ⁹⁹ ^m Tc, ¹¹¹ In, ¹³¹ I, ¹²⁵ I, ¹²⁴ I, ¹²³ I, ¹⁸⁶ Re, ¹⁸⁸ Re and ¹⁷⁷ Lu Wherein the kit is a radioisotope.

delete

The method of claim 1, wherein the chelating agent is selected from the group consisting of NOTA, DOTA, DFO, DTPA, N ₂ S ₂ , p-SCN-Bn-NOTA, NODAGA, p-SCN-Bn-DOTA, TETA, p- , p-SCN-Bn-DFO, or HYNIC.

2. The process of claim 1, wherein the Cyclooctyne group is selected from the group consisting of

,

,

,

,

,

,

or

Wherein the albumin nanoparticle preparation kit is a kit for producing albumin nanoparticles.

A solution (first solution) containing albumin monomers to which azide (N ₃ ) is bound or albumin monomers to which cyclooctane is bound;
A kit for preparing an albumin nano platform, comprising a solution (second solution) containing a delivery material to which an azide (N ₃ ) group is bonded or a delivery material to which a Cyclooctyne group is bound,
The kit is characterized in that the first solution and the second solution are subjected to a Cu-free click chemistry reaction at a pH of 6.8 to 7.6,
When the first solution is a solution containing azide (N ₃ ), the second solution is a solution containing a transfer material to which a Cyclooctyne group is bonded, and the first solution is a solution containing Cyclooctyne In the case of an included solution, the second solution is a solution containing a transfer material to which an azide (N ₃ ) group is bonded,
The albumin monomer is obtained by reacting albumin with azide (N ₃ ) or cyclooctane (Cyclooctyne) at a molar ratio of 1: 1 to 1:12,
Wherein the transmitter is a radioisotope bound to a chelating agent at a pH of 5.

delete

(a) preparing a solution in which an azide (N ₃ ) is bonded to an N-terminal amine group of an albumin monomer or a solution to which Cyclooctyne is bound; And
(b) a solution containing a cyclodextrin-bonded transfer agent or a coupling agent having an azide (N ₃ ) group bonded thereto is mixed with the solution prepared in the step (a) at a pH of 6.8 to 7.6, (Cu-free click chemistry)
When a solution in which an azide (N ₃ ) group is bound to the albumin monomer is used in the step (a), the solution is reacted with a solution containing a cyclodextrin-coupled transfer material in the step (b) When a solution in which a cyclooctyne group is bound to an albumin monomer in step a) is used, the step (b) is performed in the presence of a solution containing a conjugated azide (N ₃ )
The solution prepared in the step (a) is obtained by reacting albumin with azide (N ₃ ) or cyclooctane (Cyclooctyne) in a molar ratio of 1: 1 to 1:12,
Wherein the radioactive isotope is a radioisotope bound to the chelating agent at a pH of 5.

delete

The method of claim 9, wherein the number of the azide (N ₃ ) or the cyclooctane (Cyclooctyne) introduced into the albumin monomer is 1 to 8.

The method of claim 9, wherein the solution of step (a) is prepared by reacting the solution at a pH of 6.8 to a pH of 7.6 and a temperature of 20 to 37 캜 for 30 minutes to 1 hour.

10. The method of claim 9, wherein the Cyclooctyne group comprises