KR20130053875A

KR20130053875A - Metal nanoparticle-incorporated red blood cell and contrast agent containing the same

Info

Publication number: KR20130053875A
Application number: KR1020110119568A
Authority: KR
Inventors: 이상준; 안성숙; 정성용; 서은석
Original assignee: 포항공과대학교 산학협력단
Priority date: 2011-11-16
Filing date: 2011-11-16
Publication date: 2013-05-24

Abstract

PURPOSE: A metal nanoparticle which is surface-modified with a hydrophobic or hydrophilic functional group, and a composition containing the same for optical detection are provided to form a cluster and to enable various optical detections with surface Plasmon energy. CONSTITUTION: A red blood cell incorporated with metal nanoparticles contains red blood cells isolated from an organ and metal nanoparticles. The red blood cell contains the metal nanoparticles with a diameter of 1-100 nanometers. A method for preparing the red blood cell containing the metal nanoparticles comprises: a step of preparing the red blood cell and the metal nanoparticles; a step of treating the red blood cells and the metal nanoparticles under a hypotonic condition at 35-40 deg. C for 3-72 hours; a step of treating under an isotonic condition for 0.5-12 hours for stabilizing; a step of centrifuging the reacted material at 500-3,000rpm for 1-15 minutes and collecting a pellet; and a step of washing the pellet.

Description

Red blood cells incorporating metal nanoparticles and contrast agent containing the same {Metal nanoparticle-incorporated red blood cell and contrast agent containing the same}

The present invention provides an erythrocyte particle in which metal nanoparticles are embedded, a contrast agent including the same, and a method for producing red blood cell particles in which metal nanoparticles are embedded.

Nanotechnology is a technology that controls and controls materials at the atomic or molecular level, and is suitable for the creation of new materials or new devices, and its applications are diverse in electronics, materials, communication, machinery, medicine, agriculture, energy, and environment.

Currently, nanotechnology is developing variously and classified into three fields. First, it relates to a technology for synthesizing new materials and materials of extremely small sizes with nanomaterials. Secondly, it is a nano device and relates to a technology for manufacturing a device having a certain function by combining or arranging nano-sized materials. Third, it relates to the application of nanotechnology, called nano-bio, to biotechnology.

In particular, in the nano-bio field, nanoparticles are used for a wide range of applications such as separation of biological materials, contrast, drug / gene delivery, and the like.

Currently, iodine or barium-based contrast agents have been used to acquire X-ray images of blood vessels and digestive organs. However, the liquid contrast agent has a disadvantage in that it cannot serve as a tracer particle necessary to acquire quantitative velocity information of blood flow. Therefore, the development of contrast agents for measuring blood flow is required.

Accordingly, the present inventors have completed the present invention by incorporating metal nanoparticles having a high absorption-contrast to X-rays inside red blood cells and using high biocompatibility red blood cells themselves as tracer particles for fluid flow.

Thus, one example of the present invention provides red blood cell particles embedded with metal nanoparticles.

Another example provides a contrast agent comprising red blood cell particles in which the metal nanoparticles are embedded.

Another example provides a method of manufacturing red blood cell particles in which the metal nanoparticles are embedded.

The present invention is characterized in that the metal nanoparticles having a large absorption-contrast against X-rays are inserted into the red blood cells to use the red blood cells themselves as tracer particles of the fluid flow.

Hereinafter, the present invention will be described in more detail:

First, one example of the present invention provides red blood cell particles embedded with metal nanoparticles.

The red blood cell particles in which the metal nanoparticles are embedded include red blood cells and metal nanoparticles separated from a living body, and the metal nanoparticles are embedded in the red blood cells.

Red blood cells according to the present invention is characterized in that the metal nanoparticles with a large absorption-absorption (absorption-contrast) to the X-rays included therein, the red blood cells themselves can be used as the tracking particles of the fluid flow.

The erythrocyte is a red solid component in blood separated from a living body, preferably a human body, and may be separated from a blood sample or in the form of an artificial culture, but there is no particular limitation.

Since the metal nanoparticles must be included inside the red blood cells through the red blood cell membrane, the particle diameter is preferably 1 to 100 nanometers, and may be made of a metal material having a high absorption-contrast for X-rays. For example, the material of the metal nanoparticles may be selected from the group consisting of gold, silver, magnesium oxide, iron, platinum, titanium, alumina, zirconia, and the like. For example, chloride (III) trihydrate, HAuCl ₄ · 3H ₂ O) can be a gold nanoparticle obtained by reducing with a suitable polymer such as sodium citrate anhydride (eg, sodium citrate tribasic dehydrate), or poly (ethylene-co-propylene) copolymer. The diameter of the metal nanoparticles is not particularly limited, but is preferably 1 to 100 nm, preferably 5 to 50 nm, and more preferably about 10 to 30 nm in order to be included in the red blood cells to ensure utility as a contrast agent.

The metal nanoparticles may be variously changed in the properties of the surface while maintaining the properties of the metal nanoparticles, such as negative charge / positive charge, acid / smoke, hydrophilic / hydrophobic. The surface treatment of the metal nanoparticles determines important physical properties such as surface plasmons and can improve the encapsulation efficiency when the metal nanoparticles are introduced into red blood cells.

Thus, the metal nanoparticle may be obtained by modifying the surface with a hydrophilic or hydrophobic functional group, preferably a hydrophilic functional group. More specifically, the metal nanoparticles are

The surface modification material is introduced to the surface of the metal nanoparticles,

Particle diameter 1-100 nanometers,

The surface modification material may be an aliphatic or aromatic carboxylic acid having 1 to 20 carbon atoms, a pyrimidine base, a purine base, a straight or branched alcohol having 1 to 10 carbon atoms, and 1 to 50 carbon atoms, preferably 1 to 20 carbon atoms. It may be one or more selected from the group consisting of alkyl groups of, preferably a straight or branched alcohol having 1 to 10 carbon atoms.

The metal nanoparticles may be selected from the group consisting of gold, silver, magnesium oxide, iron, platinum, titanium, alumina, zirconia, and the like, for example, gold chloride (chloride (III) trihydrate, HAuCl ₄ · 3H ₂ O) Gold nanoparticles obtained by reduction with a suitable polymer such as sodium anhydride (eg, sodium citrate tribasic dehydrate), or poly (ethylene-co-propylene) copolymer. The diameter of the metal particles is not particularly limited, but may be 1 to 100 nm, preferably 5 to 50 nm, and more preferably about 10 to 30 nm, in order to be included in the red blood cells and to be useful as a contrast agent.

The surface modification material is introduced directly to the metal surface, thiol group, carboxyl group, amine group, aldehyde group, ketone group, peroxide group, 3 to 500 carbon atoms, preferably 3 to 100, more preferably 3 to 50, more preferably 3 to 20 alkene groups, 3 to 500 carbon atoms, preferably 3 to 100, more preferably 3 to 50, still more preferably 3 to 20 halogenated alkyl groups, ester groups, ethers It may be introduced to the metal surface through a functional group selected from the group consisting of groups, epoxide groups, nitrile groups, carbonyl groups and the like. For example, surface modification materials linked with thiol groups as functional groups for metal surface introduction include thioglycolic acid, mercaptobenzoic acid, thioguanine, mercaptoethanol, propanethiol, terphenylthiol, propenthihiol, thiazolin Thiol (Thiazolinethiol), Phenylimidazolethiol, Phenylthiazolethiol, Aminothiadiazolethiol, Bromobenzoxazolethiol, Bromopyridine fluoro benzothiol Fluorobenzoxazolethiol, Methoxybenzoxazolethiol, Carboranethiol, Menta-8-thiol-3-one, 1-4- (hydroxy Benzyl) imidazole-2-thiol [1- (4-Hydroxybenzyl) imidazole-2-thiol], 1-methyl-1 H -benzimidazole-2-thiol (1-Methyl-1 H -benzimidazole-2-thiol ), 1-phenyl -1 H - tetrazol-5-thiol (1-phenyl-1 H -tetrazole -5-thiol), 1 H -1,2,4- triazol-3-thiol (1 H -1,2,4-Triazole-3 -thiol), 3- amino-1,2,4-triazole-5-thiol (3-Amino-1,2,4-triazole -5-thiol) , 4- (trifluoromethyl) pyrimidine-2-thiol [4- (Trifluoromethyl) pyrimidine-2-thiol], 4-amino-5- (4-pyridyl) -4H-1,2,4-tria -3-thiol [4-Amino-5- (4 -pyridyl) -4 H -1,2,4-triazole-3-thiol], ( trifluoromethyl) pyrimidin-4-hydroxy-6 - 2-thiol [4-Hydroxy-6- (trifluoromethyl) pyrimidine-2-thiol], 4-methyl- 4H -1,2,4-triazole-3-thiol [4-Methyl- 4H- 1,2 , 4-triazole-3-thiol], 5- (3-pyridyl) -1,3,4-oxadiazole-2-thiol [5- (3-Pyridyl) -1,3,4-oxadiazole-2 -thiol], 5- (4-aminophenyl) -1,3,4-oxadiazole-2-thiol [5- (4-Aminophenyl) -1,3,4-oxadiazole-2-thiol], 5- (4-chlorophenyl) 1,3,4-oxadiazole-2-thiol [5- (4-Chlorophenyl) -1,3,4-oxadiazole-2-thiol], 5- (4-pyridyl)- 1,3,4-oxadiazole-2-thiol [5- (4-Pyridyl) -1,3,4-oxadiazole-2-thiol], 5-methyl-1,3,4-thiadiazole-2 -Thiol [5-Methyl-1,3,4-thiadiazole-2-thiol], 5-methylthio-1,3,4-thiadiazole-2-thiol [5 -Methylthio-1,3,4-thiadiazole-2-thiol], 5-phenyl-1,3,4-oxadiazole-2-thiol [5-Phenyl-1,3,4-oxadiazole-2-thiol] , 5-phenyl -1 H -1,2,4- triazol-3-thiol [5-phenyl-1 H -1,2,4 -triazole-3-thiol], ( trifluoromethyl) quinolin-7 4-thiol [7- (Trifluoromethyl) quinoline-4-thiol], 1- [2- (dimethylamino) ethyl] -1 H -tetrazol-5-thiol (1- [2- (Dimethylamino) ethyl]- 1 H -tetrazole-5-thiol) , 11- (1 H - pyrrole-1-yl) undecane-1-thiol [11- (1 H -pyrrol-1 -yl) undecane-1-thiol], O - (2-carboxyethyl) - O '- (2-mercaptoethyl) ethylene glycol cyclohepta [O - (2-carboxyethyl) - O' - (2-mercaptoethyl) heptaethylene glycol], O - (2- mercaptoethyl) - O '- methyl-hexa (ethylene glycol) [O - (2-Mercaptoethyl ) - O' -methyl-hexa (ethylene glycol)], O - [ ethyl-2- (3-mercapto-propionylamino)] - O '-methyl polyethylene glycol [O - [2- (3- Mercaptopropionylamino) ethyl] - O' -methylpolyethylene glycol], 1- naphthalene thiol (1-naphthalenethiol), 11- mercapto-1-undecyl kanol (11-Mercapto- 1-undecanol), 2-tee 2-Thiobarbituric acid, Cysteamine hydrochloride, Thiocholesterol, 1- (11-mercaptoundecyl) imidazole [1- (11-Mercaptoundecyl) imidazole] , Spironolactone, 1-ethyltetrazol-5-thiol, 1- (3-hydroxyphenyl) -1H-tetrazol-5-thiol [1- (3 -HYDROXYPHENYL) -1H-TETRAZOLE-5-THIOL], 1- (2-methoxyphenyl) -4- (4-nitrophenyl) 1-H-imidazole-2-thiol [1- (2-METHOXYPHENYL)- 4- (4-NITROPHENYL) -1H-IMIDAZOLE-2-THIOL], 1- (3-methylphenyl) -4- (4-methylphenyl) -1H-imidazole-2-thiol hydrobromide [1- (3-METHYLPHENYL ) -4- (4-METHYLPHENYL) -1H-IMIDAZOLE-2-THIOL HYDROBROMIDE], 1- (4- (difluoromethoxy) benzoyl) -1,4,5,6-tetrahydrocyclopenta (D) Imidazole-2-thiol [1- (4- (DIFLUOROMETHOXY) BENZOYL) -1,4,5,6-TETRAHYDROCYCLOPENTA (D) IMIDAZOLE-2-THIOL], 1- (4-aminophenyl) -4-phenyl- 1H-imidazole-2-thiol [1- (4-AMINOPHENYL) -4-PHENYL-1H-IMIDA ZOLE-2-THIOL], 1- (4-aminophenyl) tetrazol-5-thiol hydrochloride [1- (4-AMINOPHENYL) TETRAZOLE-5-THIOL HYDROCHLORIDE], 1- (4-aminophenyl) tetrazole- 5-thiol hydrochloride [1- (4-AMINOPHENYL) TETRAZOLE-5-THIOL HYDROCHLORIDE], 1-methyl-1H-imidazole-2-thiol (1-METHYL-1H-IMIDAZOLE-2-THIOL), 1-methyl -1H-tetrazol-5-thiol (1-METHYL-1H-TETRAZOLE-5-THIOL), 1-naphthalen-2-yl-1H-tetrazol-5-thiol (1-NAPHTHALEN-2-YL-1H- TETRAZOLE-5-THIOL), 1-phenyl-1H- (1,2,4) triazole-3-thiol 91-PHENYL-1H- (1,2,4) TRIAZOLE-3-THIOL], 1- (methyl Thio) -7H-pyrrolo (2,3-D) pyrimidine-4-thiol [2- (METHYLTHIO) -7H-PYRROLO (2,3-D) PYRIMIDINE-4-THIOL], 1-amino-5- (2-Chloro-phenyl) -pyrimidine-4-thiol [2-AMINO-5- (2-CHLORO-PHENYL) -PYRIMIDINE-4-THIOL], 2-methyl-9H-purine-6-thiol (2- METHYL-9H-PURINE-6-THIOL), 3-O-tolyl-6-tolyl-pyrazine-2-thiol (3-O-TOLYL-6-P-TOLYL-PYRAZINE-2-THIOL), 3-phenyl- 1,2,4-oxadiazole-5-thiol (3-PHENYL-1,2,4-OXADIAZOLE-5-THIOL), 4,5-bis (4- Methoxyphenyl) -4H-1,2,4-triazole-3-thiol (4,5-BIS (4-METHOXYPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL), 4,5-di Benzyl-4H-1,2,4-triazole-3-thiol (4,5-DIBENZYL-4H-1,2,4-TRIAZOLE-3-THIOL), 4,5-diphenyl-4H-1,2 , 4-triazole-3-thiol (4,5-DIPHENYL-4H-1,2,4-TRIAZOLE-3-THIOL), 4,6-dimethyl-pyrimidine-2-thiol (4,6-DIMETHYL- PYRIMIDINE-2-THIOL), 4- (2,3-dimethylphenyl) -5-methyl-4H-1,2,4-triazole-3-thiol [4- (2,3-DIMETHYLPHENYL) -5-METHYL -4H-1,2,4-TRIAZOLE-3-THIOL], 4- (2,4-dimethylphenyl) -5- (4-methoxyphenyl) -4H-1,2,4-triazole-3- Thiol [4- (2,4-DIMETHYLPHENYL) -5- (4-METHOXYPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4- (2,4-dimethylphenyl) -5-phenyl- 4H-1,2,4-triazole-3-thiol [4- (2,4-DIMETHYLPHENYL) -5-PHENYL-4H-1,2,4-TRIAZOLE-3-THIOL], 4- (4-bro Mophenyl) -1,3-thiazole-2-thiol [4- (4-BROMOPHENYL) -1,3-THIAZOLE-2-THIOL], 4- (4-bromophenyl) -5- (4-chloro Phenyl) -4H-1,2,4-triazole-3-thiol [4- (4-BROMOPHENYL) -5- (4-CHLOROPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4 -(4-bromophenyl) -5- (4-methoxy Phenyl) -4H-1,2,4-triazole-3-thiol [4- (4-BROMOPHENYL) -5- (4-METHOXYPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4 -(4-chlorophenyl) -1,3-thiazole-2-thiol [4- (4-CHLOROPHENYL) -1,3-THIAZOLE-2-THIOL], 4- (4-chlorophenyl) -5- ( 4-methoxyphenyl) -4H-1,2,4-triazole-3-thiol [4- (4-CHLOROPHENYL) -5- (4-METHOXYPHENYL) -4H-1,2,4-TRIAZOLE-3- THIOL], 4- (4-ethoxyphenyl) -1,5-diphenyl-1H-imidazole-2-thiol hydrobromide [4- (4-ETHOXYPHENYL) -1,5-DIPHENYL-1H-IMIDAZOLE-2 -THIOL HYDROBROMIDE], 4- (4-methoxyphenyl) -1- (4-methoxyphenyl) -1H-imidazole-2-thiol hydrobromide [4- (4-METHOXYPHENYL) -1- (4-METHYLPHENYL ) -1H-IMIDAZOLE-2-THIOL HYDROBROMIDE], 4- (benzylideneamino) -5- (2,4-dichlorophenyl) -4H-1,2,4-triazole-3-thiol [4- (BENZYLIDENEAMINO ) -5- (2,4-DICHLOROPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4- (benzylideneamino) -5- (2-bromophenyl) -4H-1,2 , 4-triazole-3-thiol [4- (BENZYLIDENEAMINO) -5- (2-BROMOPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4- (benzylideneamino) -5- ( 2-cle Rophenyl) -4H-1,2,4-triazole-3-thiol [4- (BENZYLIDENEAMINO) -5- (2-CHLOROPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4- (Benzylideneamino) -5- (2-fluorophenyl) -4H-1,2,4-triazole-3-thiol [4- (BENZYLIDENEAMINO) -5- (2-FLUOROPHENYL) -4H-1,2 , 4-TRIAZOLE-3-THIOL], 4- (benzylideneamino) -5- (2-furyl) -4H-1,2,4-triazole-3-thiol [4- (BENZYLIDENEAMINO) -5- ( 2-FURYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4- (benzylideneamino) -5- (2-methoxyphenyl) -4H-1,2,4-triazole-3 -Thiol [4- (BENZYLIDENEAMINO) -5- (2-METHOXYPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4- (benzylideneamino) -5- (2-methylphenyl) -4H- 1,2,4-triazole-3-thiol [4- (BENZYLIDENEAMINO) -5- (2-METHYLPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4- (benzylideneamino)- 5- (2-pyridinyl) -4H-1,2,4-triazole-3-thiol [4- (BENZYLIDENEAMINO) -5- (2-PYRIDINYL) -4H-1,2,4-TRIAZOLE-3- THIOL], 4- (benzylideneamino) -5- (3,4,5-trimethoxyphenyl) -4H-1,2,4-triazole-3-thiol [4- (BENZYLIDENEAMINO) -5- ( 3,4,5-TRIMETHOXYPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4- (benzyl Lidenamino) -5- (3-chlorophenyl) -4H-1,2,4-triazole-3-thiol [4- (BENZYLIDENEAMINO) -5- (3-CHLOROPHENYL) -4H-1,2,4- TRIAZOLE-3-THIOL], 4- (benzylideneamino) -5- (3-ethoxyphenyl) -4H-1,2,4-triazole-3-thiol [4- (BENZYLIDENEAMINO) -5- (3 -ETHOXYPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4- (benzylideneamino) -5- (3-isopropoxyphenyl) -4H-1,2,4-triazole-3 -Thiol [4- (BENZYLIDENEAMINO) -5- (3-ISOPROPOXYPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4- (benzylideneamino) -5- (3-methoxyphenyl)- 4H-1,2,4-triazole-3-thiol [4- (BENZYLIDENEAMINO) -5- (3-METHOXYPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4- (benzylideneamino ) -5- (3-methylphenyl) -4H-1,2,4-triazole-3-thiol [4- (BENZYLIDENEAMINO) -5- (3-METHYLPHENYL) -4H-1,2,4-TRIAZOLE-3 -THIOL], 4- (benzylideneamino) -5- (3-pyridinyl) -4H-1,2,4-triazole-3-thiol [4- (BENZYLIDENEAMINO) -5- (3-PYRIDINYL)- 4H-1,2,4-TRIAZOLE-3-THIOL], 4- (benzylideneamino) -5- (4-bromophenyl) -4H-1,2,4-triazole-3-thiol [4- (BENZYLIDENEAMINO) -5- (4-BROMOPHENYL) -4H-1 , 2,4-TRIAZOLE-3-THIOL], 4- (benzylideneamino) -5- (4-chlorophenyl) -4H-1,2,4-triazole-3-thiol [4- (BENZYLIDENEAMINO)- 5- (4-CHLOROPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4- (benzylideneamino) -5- (4-fluorophenyl) -4H-1,2,4-tria Sol-3-thiol [4- (BENZYLIDENEAMINO) -5- (4-FLUOROPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4- (benzylideneamino) -5- (4-methoxy Phenyl) -4H-1,2,4-triazole-3-thiol [4- (BENZYLIDENEAMINO) -5- (4-METHOXYPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4- ( Benzylideneamino) -5- (4-methylphenyl) -4H-1,2,4-triazole-3-thiol [4- (BENZYLIDENEAMINO) -5- (4-METHYLPHENYL) -4H-1,2,4- TRIAZOLE-3-THIOL], 4- (benzylideneamino) -5- (4-TERT-butylphenyl) -4H-1,2,4-triazole-3-thiol [4- (BENZYLIDENEAMINO) -5- ( 4-TERT-BUTYLPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4- (benzylideneamino) -5- (phenoxymethyl) -4H-1,2,4-triazole-3 -Thiol [4- (BENZYLIDENEAMINO) -5- (PHENOXYMETHYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4- (benzylideneamino) -5-cyclohexyl-4H-1,2,4 -Triazole-3-thiol [4- (BENZYLIDE NEAMINO) -5-CYCLOHEXYL-4H-1,2,4-TRIAZOLE-3-THIOL], 4- (benzylideneamino) -5-phenyl-4H-1,2,4-triazole-3-thiol [4 -(BENZYLIDENEAMINO) -5-PHENYL-4H-1,2,4-TRIAZOLE-3-THIOL], 4-allyl-5-phenoxymethyl-4H- (1,2,4) triazole-3-thiol [ 4-ALLYL-5-PHENOXYMETHYL-4H- (1,2,4) TRIAZOLE-3-THIOL], 4-amino-4H-1,2,4-triazole-3-thiol (4-AMINO-4H-1 , 2,4-TRIAZOLE-3-THIOL), 4-amino-5- (2,4-dichlorophenyl) -4H-1,2,4-triazole-3-thiol [4-AMINO-5- (2 , 4-DICHLOROPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4-amino-5- (2-bromophenyl) -4H-1,2,4-triazole-3-thiol [ 4-AMINO-5- (2-BROMOPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4-amino-5- (2-chlorophenyl) -pyrimidine-2-thiol [4-AMINO -5- (2-CHLORO-PHENYL) -PYRIMIDINE-2-THIOL], 4-amino-5- (3,4,5-trimethoxyphenyl) -4H-1,2,4-triazole-3- Thiol [4-AMINO-5- (3,4,5-TRIMETHOXYPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4-amino-5- (3-pyridinyl) -4H-1, 2,4-triazole-3-thiol [4-AMINO-5- (3-PYRIDINYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4-amino-5- (4-aminophen ) -Pyrimidine-2-thiol [4-AMINO-5- (4-AMINO-PHENYL) -PYRIMIDINE-2-THIOL], 4-amino-5- (phenoxymethyl) -4H-1,2,4- Triazole-3-thiol [4-AMINO-5- (PHENOXYMETHYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4-amino-5-butyl-4H-1,2,4-triazole 3-thiol (4-AMINO-5-BUTYL-4H-1,2,4-TRIAZOLE-3-THIOL), 4-amino-5-ethyl-4H-1,2,4-triazole-3-thiol [4-AMINO-5-ETHYL-4H-1,2,4-TRIAZOLE-3-THIOL], 4-amino-5-methyl-4H-1,2,4-triazole-3-thiol [4-AMINO -5-METHYL-4H-1,2,4-TRIAZOLE-3-THIOL], 4-benzyl-5- (2,4-dichlorophenyl) -4H-1,2,4-triazole-3-thiol [ 4-BENZYL-5- (2,4-DICHLOROPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4-benzyl-5- (3,4-dimethoxyphenyl) -4H-1,2 , 4-triazole-3-thiol [4-BENZYL-5- (3,4-DIMETHOXYPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4-benzyl-5- (4-pyridinyl ) -4H-1,2,4-triazole-3-thiol [4-BENZYL-5- (4-PYRIDINYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4-benzyl-5- (4-TERT-butylphenyl) -4H-1,2,4-triazole-3-thiol [4-BENZYL-5- (4-TERT-BUTYLPHENYL) -4H-1,2,4-TRIAZOLE-3- THIOL], 4-cyclohexyl-5- (2,4- Chlorophenyl) -4H-1,2,4-triazole-3-thiol [4-CYCLOHEXYL-5- (2,4-DICHLOROPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4- Cyclohexyl-5- (2-methoxyphenyl) -4H-1,2,4-triazole-3-thiol [4-CYCLOHEXYL-5- (2-METHOXYPHENYL) -4H-1,2,4-TRIAZOLE- 3-THIOL], 4-cyclohexyl-5- (3,4,5-trimethoxyphenyl) -4H-1,2,4-triazole-3-thiol [4-CYCLOHEXYL-5- (3,4 , 5-TRIMETHOXYPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4-cyclohexyl-5- (3,4-dimethoxyphenyl) -4H-1,2,4-triazole-3 -Thiol [4-CYCLOHEXYL-5- (3,4-DIMETHOXYPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4-ethyl-5- (4-nitrophenyl) -4H-1,2 , 4-triazole-3-thiol [4-ETHYL-5- (4-NITROPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 4-ethyl-5-M-tolyl-4H- ( 1,2,4) triazole-3-thiol [4-ETHYL-5-M-TOLYL-4H- (1,2,4) TRIAZOLE-3-THIOL], 4-ethyl-5-phenoxymethyl-4H -(1,2,4) triazole-3-thiol [4-ETHYL-5-PHENOXYMETHYL-4H- (1,2,4) TRIAZOLE-3-THIOL], 4-methyl-6-trifluoromethyl- Pyrimidine-2-thiol (4-METHYL-6-TRIFLUOROMETHYL-PYRIMIDINE-2-THIOL), 4-O-tolyl-5-P-tolyl- 4H- (1,2,4) triazole-3-thiol [4-O-TOLYL-5-P-TOLYL-4H- (1,2,4) TRIAZOLE-3-THIOL], 4-phenyl-5- (3,4,5-trimethoxyphenyl) -4H-1,2,4-triazole-3-thiol [4-PHENYL-5- (3,4,5-TRIMETHOXYPHENYL) -4H-1,2, 4-TRIAZOLE-3-THIOL], 4-phenyl-5-M-tolyl-4H- (1,2,4) triazole-3-thiol [4-PHENYL-5-M-TOLYL-4H- (1, 2,4) TRIAZOLE-3-THIOL], 5,5 '-(ethylenedithio) bis (1,3,4-thiadiazole-2-thiol) [5,5'-(ETHYLENEDITHIO) BIS (1, 3,4-THIADIAZOLE-2-THIOL)], 5,5'-tetramethylenebis (4-phenyl-4H-1,2,4-triazole-3-thiol [5,5'-TETRAMETHYLENEBIS (4-PHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL), 5,6,7,8-tetrahydro-quinazolin-2-thiol (5,6,7,8-TETRAHYDRO-QUINAZOLINE-2-THIOL) , 5,6-dihydro-4H- (1,3) thiazine-2-thiol [5,6-DIHYDRO-4H- (1,3) THIAZINE-2-THIOL], 5,7-bis (ethylamino (1,2,4) triazolo (4,3-A) (1,3,5) triazine-3-thiol [5,7-BIS (ETHYLAMINO) (1,2,4) TRIAZOLO (4, 3-A) (1,3,5) TRIAZINE-3-THIOL], 5-((1-naphthylmethyl) sulfanyl) -1,3,4-thiadiazole-2-thiol [5-(( 1-NAPHTHYLMETHYL) SULFANYL) -1,3,4-THI ADIAZOLE-2-THIOL], 5- (2,4-dichlorophenoxymethyl)-(1,3,4) oxadiazole-2-thiol [5- (2,4-DICHLORO-PHENOXYMETHYL)-(1, 3,4) OXADIAZOLE-2-THIOL], 5- (2,4-dichlorophenyl) -4- (4-methylphenyl) -4H-1,2,4-triazole-3-thiol [5- (2, 4-DICHLOROPHENYL) -4- (4-METHYLPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 5- (2,4-dichlorophenyl) -4-ethyl-4H-1,2,4 -Triazole-3-thiol [5- (2,4-DICHLOROPHENYL) -4-ETHYL-4H-1,2,4-TRIAZOLE-3-THIOL], 5- (2-chloroethylthio) -1,3 , 4-thiadiazole-2-thiol [5- (2-CHLOROETHYLTHIO) -1,3,4-THIADIAZOLE-2-THIOL], 5- (2-furyl) -4- (4-methoxyphenyl)- 4H-1,2,4-triazole-3-thiol [5- (2-FURYL) -4- (4-METHOXYPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 5- (3 -Chlorophenyl) -4- (4-fluorophenyl) -4H-1,2,4-triazole-3-thiol [5- (3-CHLOROPHENYL) -4- (4-FLUOROPHENYL) -4H-1, 2,4-TRIAZOLE-3-THIOL], 5- (3-chlorophenyl) -4-isobutyl-4H-1,2,4-triazole-3-thiol [5- (3-CHLOROPHENYL) -4 -ISOBUTYL-4H-1,2,4-TRIAZOLE-3-THIOL], 5- (3-methylphenyl) -4- (4-methylphenyl) -4H-1,2,4-triazole-3-thiol [5 -(3-ME THYLPHENYL) -4- (4-METHYLPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 5- (4-bromophenyl) -4- (2,4-dimethylphenyl) -4H-1 , 2,4-triazole-3-thiol [5- (4-BROMOPHENYL) -4- (2,4-DIMETHYLPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 5- (4- Bromophenyl) -4- (2-methylphenyl) -4H-1,2,4-triazole-3-thiol [5- (4-BROMOPHENYL) -4- (2-METHYLPHENYL) -4H-1,2, 4-TRIAZOLE-3-THIOL], 5- (4-bromophenyl) -4- (2-methylphenyl) -4H-1,2,4-triazole-3-thiol [5- (4-BROMOPHENYL)- 4- (2-METHYLPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 5- (4-chlorophenyl)-(1,3,4) oxadiazole-2-thiol [5- ( 4-CHLORO-PHENYL)-(1,3,4) OXADIAZOLE-2-THIOL], 5- (4-chlorophenyl) -pyrimidine-4-thiol [5- (4-CHLORO-PHENYL) -PYRIMIDINE-4 -THIOL], 5- (4-chlorophenyl) -4- (4-methoxyphenyl) -4H-1,2,4-triazole-3-thiol [5- (4-CHLOROPHENYL) -4- (4 -METHOXYPHENYL) -4H-1,2,4-TRIAZOLE-3-THIOL], 5- (4-chlorophenyl) -4-isobutyl-4H-1,2,4-triazole-3-thiol [5- (4-CHLOROPHENYL) -4-ISOBUTYL-4H-1,2,4-TRIAZOLE-3-THIOL], 5- (benzylthio) -1,3,4-thiadiazole-2-thiol [5- (BENZYLTHIO ) -1,3 , 4-THIADIAZOLE-2-THIOL], 5- (butylthio) -1,3,4-thiadiazole-2-thiol [5- (BUTYLTHIO) -1,3,4-THIADIAZOLE-2-THIOL], 5- (dodecylthio) -1,3,4-thiadiazole-2-thiol [5- (DODECYLTHIO) -1,3,4-THIADIAZOLE-2-THIOL], 5- (ethylthio) -1, 3,4-thiadiazole-2-thiol [5- (ETHYLTHIO) -1,3,4-THIADIAZOLE-2-THIOL], 5- (hexylthio) -1,3,4-thiadiazole-2- Thiol [5- (HEXYLTHIO) -1,3,4-THIADIAZOLE-2-THIOL], 5- (phenylthio) -1,3,4-thiadiazole-2-thiol [5- (PENTYLTHIO) -1, 3,4-THIADIAZOLE-2-THIOL], 5-amino-1,3,4-thiadiazole-2-thiol [5-AMINO-1,3,4-THIADIAZOLE-2-THIOL], 5-amino- 4-phenyl-4H- (1,2,4) triazole-3-thiol [5-AMINO-4-PHENYL-4H- (1,2,4) TRIAZOLE-3-THIOL], 5-benzyl-4- Phenyl-4H- (1,2,4) triazole-3-thiol [5-BENZYL-4-PHENYL-4H- (1,2,4) TRIAZOLE-3-THIOL], alkanes having 3 to 500 carbon atoms and carbon atoms It may be one or more selected from the group consisting of 3 to 500 alkenthiol and the like.

Metal nanoparticles modified with these surface modifying materials have a uniform diameter of about 20 nm, and the particle spacing and cluster size are suitable as X-ray contrast agents when forming clusters in a suitable medium. In addition, the erythrocyte particles may be used as a tracer for X-ray imaging, which is necessary to measure the velocity distribution of blood flow as a particle contrast agent in place of a liquid contrast agent.

The amount of the metal nanoparticles inherent in one red blood cell is not particularly limited, but may be included in an amount capable of forming aggregates having an average diameter of 1 to 3 μm in the red blood cells in order to be detectable and used as a contrast agent.

In addition, the gold nanoparticles according to the present invention have surface plasmon energy not only in the X-ray region but also in the visible region, thereby enabling detection in the visible region and the UV region (see Example 4 and FIGS. 5A to 5E). .

The red blood cell particles in which the metal nanoparticles are embedded may be added to the red blood cells by adding a metal nanoparticle, preferably a hydrophilic / hydrophobic surface-modified metal nanoparticle solution, and then placing the metal nanoparticle in a storage (hypotonic) condition. It may be obtained by inducing nanoparticles to enter the red blood cells, stabilizing in isotonic conditions after a certain time and centrifuging to separate and wash the red blood cell-gold particles (see FIG. 1).

As described above, the red blood cell particles incorporating the metal nanoparticles, preferably the surface-modified metal nanoparticles, according to the present invention are useful as contrast agents, such as optical detection contrast agents. It provides a contrast agent containing a red blood cell inherent.

The contrast agent comprises red blood cells containing the metal nanoparticles in water, 3 to 500 carbon atoms, preferably 3 to 100 carbon atoms, more preferably 3 to 50 carbon atoms, more preferably 3 to 20 linear or branched alcohols, carbon atoms 3 to 500, preferably 3 to 100, more preferably 3 to 50, more preferably 3 to 20 aldehydes, 3 to 500 carbon atoms, preferably 3 to 100, more preferably 3 to 50, further 100 ppm to 10 wt%, preferably 100 to 10,000, in one or more solvents (or media) selected from the group consisting of ketones of 3 to 20, solvents of normal paraffins of 5 to 20 carbon atoms, and the like. ppm, more preferably 100 to 1,000 ppm.

The normal paraffinic solvents are those having a boiling point of 40 ° C. or lower, for example, n-pentane, and isomers thereof (eg, isopentane, neopentane, etc.), hexane, methylpentane (eg, 2-methylpentane, 3- Methylpentane, etc.), methylbutane (eg, 2,3-dimethylbutane, 2,2-dimethylbutane, etc.), n-heptane and its isomers (eg 2-methylhexane (isoheptane), 3-methylhexane, 2,2-dimethylpentane (neoheptane), 2,3-dimethylpentane, 2,4-dimethylpentane, 3,3-dimethylpentane, 3-ethylpentane, 2,2,3-trimethylbutane, etc.), octane, n-octane and its isomers (eg 2-methylheptane, 3-methylheptane (2 ethanethiomers), 4-methylheptane, 3-ethylhexane, 2,2-dimethylhexane, 2,3-dimethylhexane (2 enantiomers), 2,4-dimethylhexane (2 enantiomers), 2,5-dimethylhexane, 3,3-dimethylhexane, 3,4-dimethylhexane (2 enantiomers + 1 Meso compound, 3-ethyl-2-methylpentane, 3-ethyl-3-methylpentane, 2,2,3-trimethylpentane (2 Enantiomers), 2,2,4-trimethylpentane (isooctane), 2,3,3-trimethylpentane, 2,3,4-trimethylpentane, 2,2,3,3-tetramethylbutane), n -Nonane and its isomers, n-decane and its isomers, n-undecane and its isomers, n-dodecane and its isomers, n-tridecane and its isomers, n-tetradecane and its isomers , n-pentadecane and its isomer, n-hexadecane and its isomer, n-heptadecane and its isomer, n-octadecane and its isomer, n-nonadecan and its isomer, n-eicoic acid And it may be one or more selected from the group consisting of isomers and the like.

As described above, the red blood cell particles embedded in the metal nanoparticles according to the present invention are included in the solvent in the visible range of the embedded metal nanoparticles, preferably the surface-modified metal nanoparticles. Since surface plasmon energy is easily detected, the solvent (or medium) and the concentration of the red blood cell particles incorporating the metal nanoparticles are preferably within the above range. In a preferred embodiment, the red blood cell particles embedded in the metal nanoparticles according to the present invention are 100 ppm to 10 wt%, preferably 100 to 10,000 ppm, more preferably 100 to 1,000 ppm, such as 300 to 700 ppm, in an aqueous solution. It can be injected into the body at a concentration of.

The contrast agent according to the present invention can be usefully used for measuring blood flow, and can be used for X-ray imaging. For example, the contrast agent quantitatively analyzes the flow information of the blood flow in vivo using diagnostic defenses such as angiography, ultrasound, MRI, X-ray imaging (including both clinical X-rays and X-ray radiation) and PIV velocity field measurement techniques. It can be a new concept of contrast agent that can be used for flow analysis and early diagnosis of cardiovascular disease.

Specifically, the contrast agent may be applied to all imaging areas using X-rays such as X-ray CT, angiography, and X-ray imaging technology of a radiation accelerator. X-rays have been widely used for medical diagnosis and non-destructive testing because they easily penetrate opaque living bodies or objects. In particular, with the recent development of radiation accelerators and digital image processing techniques, it is possible to obtain images of living biological samples with high spatial resolution and excellent contrast. As a result, various imaging techniques using X-rays have been developed and their application is expanding to many fields such as life sciences, medical engineering, and materials engineering. X-ray PIV, which can measure flow velocity information on the flow of opaque fluid like blood Technique is one of them. The PIV (Particle Image Velocimetry) technique is a quantitative flow visualization technique that is widely used in the field of hydrodynamics in recent years. Velocity field information is obtained by applying a digital image processing technique to a flow image containing tracer particles. Using PIV technique, we can extract the entire velocity field information of the fluid flow instantaneously by applying velocity field extraction algorithms such as cross-correlation to two particle images acquired at short time intervals (Δt). have.

Early diagnosis of circulatory vascular diseases, which is the leading cause of death in modern people, is very important, and the present invention is an essential technology for developing a new image diagnosis technology that can contribute to it. When the contrast agent containing the red blood cell particles embedded with the metal nanoparticles of the present invention is applied to the X-ray PIV technique, the flow information may be measured using tracer particles flowing along opaque blood. Therefore, the contrast agent of the present invention can measure the overall velocity field distribution of blood flow instantaneously, which will greatly contribute to the early diagnosis of cardiovascular disease in the future.

Another example of the present invention provides a method for producing red blood cell particles in which the metal nanoparticles are embedded. The method

Preparing red blood cells and metal nanoparticles separated from the living body;

Treating the red blood cells and the metal nanoparticles at a storage condition of 35 to 40 ° C. for 3 to 72 hours, and preferably 3 to 24 hours to insert the metal nanoparticles into the red blood cells;

Treating the stored red blood cells and metal nanoparticles under isotonic conditions for 0.5 to 12 hours to stabilize them;

Centrifuging the reaction product at a speed of 500 rpm to 3000 rpm for 1 to 15 minutes to remove the supernatant and take pellet; And

Washing the obtained pellets

It may include (see Figure 2).

The red blood cells and the metal nanoparticles are as described above.

The metal nanoparticles may be used in an amount of 0.25-4 volume times, more specifically 0.5-2 volume times, of the red blood cell volume, based on the case where the solution is used as a 2.4 × 10 ¹⁸ AuNPs / m ³ concentration solution. For example, red blood cells may be used in an amount of 2 to 0.5 mL and metal nanoparticles may be used in an amount of 0.25 to 4 volume times, more specifically 0.5 to 2 volume times, based on the concentration of 2.4x10 ¹⁸ AuNPs / m ^3. have.

The storage condition means a concentration environment lower than the red blood cell osmotic pressure, and the treatment step in the storage condition may be performed in all solutions having a concentration lower than the red blood cell osmotic pressure which can form such a storage environment. For example, the treatment step in the storage conditions may be a buffer solution (such as PBS, etc.) in which the concentration of deionized water or ions is lower than the red blood cell osmotic pressure (0.9 to 1.1% (w / v)), and the pH is It can be fully deionized and neutral, or adjusted to pH 7-8, specifically about pH 7.2-7.6, specifically about pH 7.4, and conditions of acid or base are preferably avoided. For example, for storage conditions a buffer solution diluted with deionized water or deionized water to a concentration lower than 0.9% (w / v), for example 0.5% (w / v) or less, can be used.

The treatment step in the storage condition is preferably performed for 3 to 72 hours, preferably 3 to 24 hours. If the reaction time is shorter than the above range, the metal nanoparticles are not sufficiently introduced into the red blood cells, and after about 24 hours, The number of incoming metal nanoparticles is almost saturated and remains substantially constant, and after about 72 hours, the processing time in storage conditions becomes longer and the low concentration stress of erythrocytes becomes severe. good.

The isotonic condition means a concentration environment equivalent to erythrocyte osmotic pressure, and the treatment step in the isotonic condition is pH 7 to 8, specifically, the concentration is adjusted to an erythrocyte osmotic pressure range, specifically 0.9 to 1.1% (w / v). all solutions with a pH of 7.2 to pH 7.6, such as about pH 7.4. For example, the treatment step in the isotonic condition may be performed at pH 7 to 8, specifically pH 7.2 to pH 7.6, such as about pH 7.4, in which the ion concentration is adjusted to an erythrocyte osmotic pressure range, specifically 0.9 to 1.1% (w / v). Buffer solutions and the like may be used, and acid or base conditions are preferably avoided.

The washing step is washing with a buffer solution (eg PBS buffer), water (eg distilled water, deionized water), diluent (eg, mixing PBS with water) and the like, for example pH 7 to 8, specifically pH 7.2 to 7.6, more specifically about pH 7.4 buffer solution, distilled or deionized water, and pH 7 to 8, specifically pH 7.2 to 7.6, more specifically about pH 7.7 buffer solution to be performed in order Can be.

A new type of contrast medium of the present invention will be utilized to measure invisible opaque in vivo blood flow at a resolution of several micrometers and to measure in real time the rate field change of blood flow over time. This will mark a milestone in the medical field as well as early diagnosis of circulatory diseases.

FIG. 1 schematically shows gold nanoparticles (AuNP1, AuNP2, AuNP3, AuNP4, AuNP5, and AuNP6) prepared in Example 1. FIG.
FIG. 2 schematically shows how to insert gold nanoparticles into red blood cells in Example 1.2.
3A to 3C show SEM and EDS images of red blood cells embedded with gold nanoparticles AuNP 1 (3a), AuNP 5 (3b), and AuNP6 (3c), respectively, prepared in Example 1;
3d is a TEM image of RBC-AuNPs,
3e is an X-ray image of RBC 0, RBC 4, RBC 5, and RBC 6.
Figure 4 shows the change of X-ray image with time of erythrocytes embedded with gold nanoparticles AuNP 5 (A) and AuNP 6 (B), respectively.
Figure 5a shows the size of the gold nanoparticles prepared by the method of triblock copolymer polymer (Pluronic) formed of poly (ethylene-co-propylene) to reduce the gold ions to gold nanoparticles, (A) Pluronic P84 10wt It is reduced gold nanoparticles at% conditions, (B) is a gold nanoparticles reduced at 30wt% of Pluronic P84, (C) shows the gold nanoparticles reduced at 50wt% of Pluronic P84 ,
5b shows that when the amphiphilic polymer Pluronic 84 is introduced into a solution dissolved in water, 5b has a surface plasmon energy in the visible region.
5c is a graph showing the results of measurement of surface plasmon energy of particles shown in 5b by UV-vis spectroscopy,
5D shows hydrophilic gold nanoparticles AuNP5 in the gold nanoparticles shown in FIG. 1 shows a unique color when the amphiphilic polymer Pluronic 84 is introduced into a solution dissolved in water, and has surface plasmon energy in the visible region. Showing,
5e shows hydrophobic gold nanoparticles. AuNP 6 in the gold nanoparticles shown in FIG. 1 shows a unique color when the amphiphilic polymer Pluronic 84 is introduced into a solution dissolved in water, and has surface plasmon energy in the visible region. (The numbers 0.1 to 0.9 described in the test tubes of FIGS. 5B, 5D and 5E represent concentrations of Pluronic 84, where 0.1 is 10% by weight, 0.2 is 20% by weight, 0.3 is 30% by weight, and 0.4 is 40). Weight percent, 0.5 means 50 weight percent, 0.6 means 60 weight percent, 0.7 means 70 weight percent, 0.8 means 80 weight percent and 0.9 means 90 weight percent).

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1: gold nanoparticles Inherent Manufacture of red blood cells

1.1: Preparation of Gold Nanoparticles

Gold chloride (III) trihydrate (HAuCl ₄ · 3H ₂ O) was dissolved in de-ionized water to make a solution of 1.0 x 10 ^-3 mol / L, and 200 mL of this solution was dissolved in sodium citrate tribasic dehydrate. The surface of the gold particles was reduced by adding 20 mL of a solution dissolved in the solution to a concentration of 4 × 10 ⁻² mol / L and refluxing. After maintaining the reflux state for 30 minutes, the temperature was lowered to 25 ℃ to adjust the size of the particles to about 20nm. When the particles are completed after the reduction reaction, Spectra / Por

The membrane (1,000 Da cut) was dialyzed overnight in deionized water to remove unreacted impurities.

After reducing as described above, the particles that did not additionally react with the molecule was named AuNP 1. In addition, 40 mL of 0.1M thioglycolic acid (SH-CH ₂ COOH) in 10 mL of a solution in which the reduced particles were dissolved in a concentration of 2.4 x 10 ¹⁸ AuNPs / m ³ was dissolved in deionized water, and 0.1M 4-mercapto. 40 mL benzoic acid (4-mercaptobenzoic acid, SH-Ph-COOH), 40 mL 0.1 M 6-thioguanine (SH- C ₅ H ₄ N ₅ ), 0.1 M 2-mercaptoethanol (SH) 40 mL of -CH ₂ CH ₂ OH) and 40 mL of 0.1M 1-propanehiol (1-propanthiol, SH-CH ₂ CH ₂ CH ₃ ) were added at room temperature, respectively, and then maintained at 50 to 60 ° C. for 6 to 12 hours. The reaction was carried out until no further color change. Spectra / Por when the reaction is complete

7 membrane (1,000 Da cut) was dialyzed overnight in deionized water to remove unreacted impurities. The particles thus obtained were respectively AuNP 2 (modified with SH-CH ₂ COOH), AuNP 3 (modified with SH-Ph-COOH), AuNP 4 (modified with SH- C ₅ H ₄ N ₅ ), AuNP 5 (SH- Modified with CH ₂ CH ₂ OH), and AuNP (modified with SH—CH ₂ CH ₂ CH ₃ ) (see FIG. 1).

1.2: gold nanoparticles Inherent Manufacture of red blood cells

Blood (Daegu Gyeongbuk Blood Source) was centrifuged at 1500 rpm for 5 minutes to remove supernatant and red blood cells. To 1 mL of the isolated red blood cells, a standard solution (2.4 × 10 ¹⁸ AuNPs / m ³ ) of the gold nanoparticles prepared in Example 1.1 was added in an amount of 1 mL to be 1 volume of the red blood cell volume, respectively. The samples thus prepared were stored under hypotonic conditions (deionized water at pH 7.4) at 37 ° C. for a defined time (3 to 72 hours). After stabilizing in PBS buffer for about 10 minutes, the resultant was washed with deionized water to prevent salts from remaining. The samples were stored and stabilized for 1 hour in isotonic conditions (PBS buffer solution of pH 7.4: NaCl 137 mM, KCl 2.7 mM, Na ₂ HPO ₄ 4.3 mM, KH ₂ PO ₄ 1.4 mM in 1000 ml), and then at 2700 rpm. The supernatant was removed by centrifugation for 5 minutes, and pellets were taken to separate erythrocyte-gold particles. The separated particles were washed in the order of PBS buffer solution of distilled water-pH 7.4 at pH 7.4 to PBS buffer solution to finally obtain red blood cells (RBC-AuNPs) containing gold nanoparticles (see FIG. 2).

In order to confirm the time condition as described above, various results were obtained while changing the storage condition treatment time from 3 hours to 72 hours, and the obtained results are shown in FIG. 4. As can be seen in Figure 4, the results of entering the gold nanoparticles vary depending on the storage conditions treatment time. More specifically, FIGS. 4A and 4B show nanoparticles entering red blood cells in RBC 5 containing hydrophilic gold nanoparticles (AuNP 5) and RBC 6 containing hydrophobic gold nanoparticles (AuNP 6), respectively. As can be seen, there is a difference in the way these particles enter the red blood cells. In other words, the hydrophilic gold nanoparticles are shown in a reversible manner, it can be seen that the hydrophobic gold nanoparticles are introduced into the red blood cells in an irreversible process. 4 (C) shows the results of UV-vis spectroscopy (HP, HP8453), which is the physical property of such gold nanoparticles, and confirms the above-mentioned contents again. 4D shows the result of hemoglobin flowing out as a result of the introduction of the gold nanoparticles. It can be seen that the results are varied depending on the treatment time and the concentration of gold nanoparticles. It can also be observed that the results vary with time and concentration depending on the type of surface modified material.

Example 2: SEM ( Scanning Electron Microscope ), And EDS ( Energy Disperse X-ray Spectrometer )

Gold nanoparticles AuNP 1, AuNP 5, or AuNP6-containing red blood cells (RBC-AuNP1, RBC-AuNP5, and RBC-AuNP6) prepared in Example 1 were used at 15 kV using SEM (JEOL JSM-7401F SEM). X-rays were taken on a single red blood cell taken with a micro-scale photograph, and SEM was used to select the exact spot (RBC region) to know the composition from the SEM photographs and analyzed by EDS (JEOL JSM-7401F SEM). The weight ratio and atomic number ratio of gold to carbon and oxygen were calculated. The obtained results are shown in FIGS. 3A to 3C. It can be seen that gold is introduced into the red blood cells.

Example 3: TEM ( Transmission electron microscopy )

Karnovsky's fixed solution (2% (w / v) paraformaldehyde and 2% (w / v) glutaraldehyde in 0.05 M sodium cacodylate buffer at pH 7.2) modified with red blood cells (RBC-AuNPs) containing gold nanoparticles prepared in Example 1 ) Was first fixed at 4 ° C. for 2-4 hours. The sample was washed three times with 0.05 M sodium cacodylate buffer (pH 7.2) for 10 minutes at 4 ° C. The 1% osmium tetroxide solution dissolved in 0.05 M sodium cacodylate buffer (pH 7.2) was used for secondary fixation at 4 ° C. for 2 hours. After washing twice more with distilled water at room temperature, en bloc staining was performed for 30 minutes at 4 ° C using 0.5% (w / v) uranyl acetate. Samples were dehydrated at room temperature for 10 minutes and at each stage 30% (v / v), 50% (v / v), 70% (v / v), 80% (v / v), 90% (v / v) ), 100% (v / v), 100% (v / v), and 100% (v / v) ethanol. The transition was performed at room temperature for 15 minutes using 100% (w / v) propylene oxide. Propylene oxide: Spurr's resin ratios of 2: 1 (for 1 h), 1: 1 (for 1 h), 1: 2 (for 2 h), 0: 1 (for 4 h), and 0: 1 (for 2 h) mixture was treated for the stated time to penetrate the sample. The polymerization was carried out at 70 ° C. for 24 hours. After slicing the obtained sample (RBC-AuNPs) using ultramicrotome (MT-X, RMC, Tucson, AZ, USA), for 2 minutes with 2% (w / v) uranyl acetate and 2 minutes with Reynolds' lead citrate Stained. TEM images were captured using JEM-1011 (JEOL, Tokyo, Japan).

TEM images of the obtained RBC-AuNPs are shown in FIG. 3D. 'RBC 0' in the figure means that the red blood cells were treated only with deionized water without AuNP treatment. Erythrocyte cells containing AuNP 1, AuNP 2, AuNP 3, AuNP 4, AuNP 5, or AuNP 6 were designated as RBC 0, RBC 2, RBC 3, RBC 4, RBC 5 and RBC 6, respectively.

Example 4: X-ray imaging

As shown in Example 1.2, the image obtained when the erythrocytes and AuNP 5 and AuNP 6 were put together for 3, 6, 12 and 24 hours under hypotonic conditions using an 1B2 and 7B2 beamline (Pohang Accelerator Laboratory) X-ray images were taken. The obtained X-ray image is shown in FIG. 4. In FIG. 4, the contrast is colored to show a large contrast according to the X-ray absorption rate in order to increase the contrast. As can be seen in FIG. 4, when 3 hours have elapsed, the X-ray absorption effect of both kinds of particles is not large. On the other hand, after 6 hours, X-ray absorption increased significantly. In the case of AuNP 6, the X-ray absorption increased continuously after 6 hours, but in the case of AuNP 5, the absorption was decreased at 12 hours. Increased again at 24 hours. Hydrophilic gold nanoparticles are introduced into and out of the red blood cells through the channel through which the water of the red blood cells moves, but once the hydrophobic gold nanoparticles are introduced into the red blood cells, they are introduced as an irreversible process.

In addition, X-ray images of the selected RBC 0, RBC 4, RBC 5, and RBC 6 are shown in Figure 3e. Since the X-ray images of RBC 1, RBC 2, and RBC 3 are almost similar to RBC 0, RBC 0, RBC 4, RBC 5, and RBC 6 were compared. The upper one shows the absolute scale (I) and the lower one shows the relative intensity by background subtraction (II ₀ ). The darker the color in the image, the higher the X-ray absorption.

Example 5: Surface modified Surface in the visible region of gold nanoparticles Plasmon Energy check

Gold nanoparticles were prepared by mixing a gold salt (chloride (III) trihydrate, HAuCl ₄ · 3H ₂ O, 0.5 g / 200 mL water) with Pluronic 84.

More specifically, the gold salt aqueous solution (0.5g / 200mL water) and Pluronic 84 is 0.9: 0.1, 0.8: 0.2, 0.7: 0.3, 0.6: 0.4, 0.5: 0.5, 0.4: 0.6, 0.3: 0.7, 0.2 Gold nanoparticles were prepared by mixing gold salts by mixing at a weight ratio of: 0.8, and 0.1: 0.9 (weight of aqueous gold salt solution: weight of Pluronic 84).

Among them, the weight ratio of the aqueous gold salt solution and Pluronic 84 was obtained under the conditions of 0.9: 0.1 [FIG. 5A (A)], 0.7: 0.3 [FIG. 5A (B)], and 0.5: 0.5 [FIG. 5A (C)]. The appearance of the gold nanoparticles is shown in Figure 5a.

In addition, the color change when the nanoparticles are prepared by reducing the gold salt with Pluronic 84 at various concentrations is shown in FIG. 5B. The numerical value shown in FIG. 5B shows the weight ratio of Pluronic 84 when the total weight of the gold salt aqueous solution and Pluronic 84 mixture is set to 1. FIG. As shown in FIG. 5B, when gold nanoparticles are introduced into a solution in which amphiphilic polymer Pluronic 84 is dissolved in water, it shows a unique color according to the polymer concentration, which shows that it has surface plasmon energy in the visible region. .

In addition, surface plasma surface energy at each of these concentrations was measured using a UV-vis spectrometer (HP, HP8453), and the results are shown in FIG. 5C. As shown in FIG. 5C, when the weight ratio of Pluronic 84 is 0.1 and 0.9, a distinct peak is observed to be weak, indicating that the formation of particles is not apparent or that there is no particle formation in the measured energy region. Under conditions where the weight ratio of RONIC 84 is from 0.2 to 0.8, gold nanoparticles showing surface plasmon energy were formed in the measured area.

AuNP 2 modified with hydrophilicity (SH-CH ₂ CH ₂ OH) among the gold nanoparticles prepared in Example 1 was represented by AuNP 5 in FIG. The color change was observed in FIG. 5D. The numerical values in FIG. 5D are as described in FIG. 5B at the concentration of Pluronic 84 aqueous solution. As shown in FIG. 5D, it can be seen that the hydrophilically modified gold nanoparticles in accordance with the present invention exhibit unique surface plasmons in the visible region within the specific microstructures produced by Pluronic 84 at various concentrations.

Among the gold nanoparticles presented in Example 1, AuNP 3 (denoted as AuNP 6 in FIG. 5E) modified with hydrophobicity (SH-CH ₂ CH ₂ CH ₃ ) was added to Pluronic 84 aqueous solution of various concentrations as described above. The change in color was observed and shown in FIG. 5E. The numerical values in FIG. 5E are as described in FIG. 5B at the concentration of Pluronic 84 aqueous solution. As shown in FIG. 5E, it can be seen that the hydrophilically modified gold nanoparticles according to the present invention exhibit unique surface plasmons in the visible region within the specific microstructures produced by various concentrations of Pluronic 84.

Claims

Comprising red blood cells and metal nanoparticles isolated from a living body,
Characterized in that the metal nanoparticles having a particle diameter of 1 to 100 nanometers embedded in the red blood cells,
Red blood cell particles embedded with metal nanoparticles.

The method of claim 1, wherein the metal nanoparticles are selected from the group consisting of gold, silver, magnesium oxide, iron, platinum, titanium, alumina, and zirconia,
Red blood cell particles embedded with metal nanoparticles.

The method of claim 1, wherein the metal nanoparticles,
The surface modification material is introduced to the surface of the metal particles,
The surface modifier is selected from the group consisting of aliphatic or aromatic carboxylic acids having 1 to 20 carbon atoms, pyrimidine bases, purine bases, straight or branched alcohols having 1 to 10 carbon atoms, and alkyl groups having 1 to 50 carbon atoms. More than one species,
Red blood cell particles embedded with metal nanoparticles.

The method of claim 3,
The surface modification material may be a thiol group, a carboxyl group, an amine group, an aldehyde group, a ketone group, a peroxide group, an alken group having 3 to 500 carbon atoms, a halogenated alkyl group having 3 to 500 carbon atoms, an ester group, an ether group, or an epoxide group. Is introduced to the metal surface through a functional group selected from the group consisting of, nitrile group, and carbonyl group,
Red blood cell particles embedded with metal nanoparticles.

The method of claim 3,
The surface modification material is at least one member selected from the group consisting of linear or branched alcohols having 1 to 10 carbon atoms,
Red blood cell particles embedded with metal nanoparticles.

A contrast agent comprising an erythrocyte particle embedded with a metal nanoparticle according to any one of claims 1 to 5.

According to claim 6, wherein the red blood cell particles in which the metal nanoparticles are embedded, water, linear or branched alcohol of 3 to 500 carbon atoms, aldehyde of 3 to 500 carbon atoms, ketone of 3 to 500 carbon atoms, and normal of 5 to 20 carbon atoms Contrast agent comprising a concentration of 100 ppm to 10 wt% in at least one solvent selected from the group consisting of paraffin-based solvents.

The contrast agent of claim 6, wherein the contrast agent is for use in measuring blood flow.

The contrast agent of claim 6, wherein the contrast agent is for use in X-ray contrast, ultrasound, angiography, MRI, or particle imaging flow meter (PIV) measurements.

Preparing red blood cells and metal nanoparticles separated from the living body;
Treating the red blood cells and the metal nanoparticles at a storage condition of 35 to 40 ° C. for 3 to 72 hours to insert the metal nanoparticles into the red blood cells;
Treating the stored red blood cells and metal nanoparticles under isotonic conditions for 0.5 to 12 hours to stabilize them;
Centrifuging the reaction product at a speed of 500 rpm to 3000 rpm for 1 to 15 minutes to remove the supernatant and take pellet; And
Washing the obtained pellets
Claims 1 to 5, wherein the metal nanoparticles of any one of claims, wherein the manufacturing method of red blood cells embedded.

The method of claim 10,
The washing step is performed by sequentially washing the buffer solution of pH 7 to 8, distilled water, and the buffer solution of pH 7 to 8,
Gt;