KR20100037734A - Bioavailable quantum dots as a molecular optical imaging agent and preparation method thereof - Google Patents

Abstract

PURPOSE: A method for preparing quantum dot for biocompatible molecular optical image is provided to enhance circulation time in blood and to reduce side effect by toxicity and accumulation in an organism. CONSTITUTION: A quantum dot for biocompatible molecular image comprises cysteamine and soluble monosaccharide bound on the surface of quantum dot comprising cadmium selenide core and zinc sulfide shell. The soluble monosaccharide is gluconic acid, citric acid, propionic acid, butyric acid, or oleic acid. A method for producing the quantum dot comprises: a first step synthesizing a quantum dot comprising cadmium selenide core and zinc sulfide shell; a step of introducing cysteamine on the surface of the quantum dot to modifying the surface of the quantum dot to hydrophilic; and a step of introducing soluble monosaccharide to the modified quantum dot.

Description

Bioavailable quantum dots as a molecular optical imaging agent and preparation method

The present invention relates to a quantum dot for biocompatible molecular optical imaging and a method of manufacturing the same.

A quantum dot is a kind of light emitting semiconductor device made of inorganic material, and its material is generally different bandgap such as silver sulfide (Ag ₂ S), cadmium sulfide (CdS), cadmium selenium (CdSe), or titania (TiO 2). It is a conjugate of heterogeneous materials having a size of 5 to 15 nm. Quantum dot manufacturing techniques are well established from the late 1990s to the early 2000s. Quantum dots, unlike conventional lumped semiconductors, exhibit a variety of unique optical, electrical and magnetic properties depending on the size, shape, or composition of the particles, and are excellent in stability. Generally, by coating a conjugate of a heterogeneous material having another bandgap on the surface of a quantum dot made of a conjugate of a heterogeneous material, the quantum yield is amplified and modified to exhibit hydrophilicity by surface modification using a polymer. I use it.

In Korea, researches are being made on magnetic resonance imaging (MRI) by using nano-sized magnetic particles at mechanical research institutes, ceramics research institutes, and chemical research institutes. Although attempts have been made to detect quantum dots, few studies have been attempted to apply quantum dots as medical contrast agents using optical images.

In order to increase the application of quantum dots in the field of biomolecular imaging, the surface of the synthesized nanoparticles should be treated to be biocompatible, mainly silica, polyethyleneglycol (PEG), glutathione, cysteamine, mercaptoscuccinic By introducing acid into the surface, studies are being actively conducted to change the degree of hydrophilicity as well as the distribution in vivo. However, modification of these surfaces is limited in in vivo studies. Most surface modified quantum dots are meaningful only in vitro studies and have limitations in in vivo studies.

In addition, Cho et al. (Long-term exposure to CdTe quantum dots causes functional impairments in live cells, Langmuir, 23, 1974-1980, 2007) prepared hydrophilic quantum dots using various surface modifiers and used them in cell experiments, Hoshino et al. (Quantum dots targeted to the assigned organelle in living cells, Microbiol. Immunol. 48, 985-994, 2004) were also used for cell experiments using quantum dots with carboxy groups and quantum dots with amine groups. There are no published articles describing the results of in vivo experiments.

Surface modification of quantum dots using materials with amine groups, such as cysteamine, can increase hydrophilicity, introduce target ligands such as antibodies, and disperse well in precipitates in weakly acidic waters, but accumulate in certain organs. The blood circulation time in vivo is so short that there are many limitations for in vivo studies.

In order to overcome this problem, the present inventors have introduced cysteamine into quantum dots and introduced gluconic acid to solve the toxicity problem and short blood circulation time that accumulate in certain organs in vivo by cysteamine. It has been confirmed that the present invention has been completed.

An object of the present invention is to provide a quantum dot for biocompatible molecular imaging, characterized in that the cysteamine and a water-soluble monosaccharide are bonded to the surface of the quantum dot consisting of a cadmium selenide core and zinc sulfide shell.

In addition, another object of the present invention is to introduce a cysteamine on the surface of the quantum dot consisting of a cadmium selenide core and a zinc sulfide shell to modify the surface of the quantum dot to be hydrophilic, and to introduce a water-soluble monosaccharide to the surface-modified quantum dot It is to provide a method of manufacturing a quantum dot for biocompatible molecular imaging prepared by.

In order to achieve the above object, the present invention provides a quantum dot for biocompatible molecular imaging, characterized in that the cysteamine and a water-soluble monosaccharide are bonded to the surface of the quantum dot consisting of a cadmium selenide core and a zinc sulfide shell.

In addition, the present invention comprises a first step of synthesizing a quantum dot consisting of a cadmium selenide core and a zinc sulfide shell; Introducing a cysteamine to the surface of the synthesized quantum dots to modify the surface of the quantum dots hydrophilically; And a third step of introducing a water-soluble monosaccharide into the hydrophilic surface-modified quantum dot.

Hereinafter, the present invention will be described in detail.

According to the first aspect of the present invention, the present invention provides a quantum dot for biocompatible molecular imaging, characterized in that cysteamine and a water-soluble monosaccharide are bonded to a surface of a quantum dot made of a cadmium selenide core and a zinc sulfide shell. In addition to cysteamine, a material having a thiol group and an amine group may be used for surface modification of the quantum dots.

In the present invention, the surface modification of the quantum dot using a material having an amine group, such as cysteamine represented by the following formula (1) can increase the hydrophilicity, introduce a target ligand such as an antibody, and in weakly acidic water It can be dispersed well without precipitation.

 Cysteamine

In the present invention, the water-soluble monosaccharide can be selected from the group consisting of gluconic acid, citric acid, propionic acid, butyric acid and oleic acid, the size of the quantum dot is increased by binding the water-soluble monosaccharide to the surface of the hydrophilic quantum dots, macrophages It can improve the blood circulation reduction time by. In one embodiment of the present invention, gluconic acid having the following formula was selected.

Gluconic Acid

Quantum dots with cysteamine introduced mainly in the lungs are highly toxic in vivo, but the introduction of gluconic acid not only changes the distribution in vivo, but also can be rapidly excreted in vivo to minimize accumulation. The toxicity of heavy metals can be minimized. That is, gluconic acid improves blood circulation time reduction by macrophages, thereby reducing intake by Cooper cells, and increases blood circulation time. Also, when excessively treated, gluconic acid cannot bind antibodies or bind very small amounts. Since there may be a problem, preferably, the gluconic acid and the hydrophilic surface-modified quantum dot are combined at a weight ratio of 1: 1 to 30.

In one embodiment of the present invention, the blood circulation time in the gluconic acid-bound quantum dot nanoparticles is extended and is mainly discharged through the kidney, it can be seen that the amount discharged through the kidney increases with the increase in the amount of gluconic acid there was.

The present invention also relates to quantum dots for biocompatible molecular imaging in which an antibody or peptide for cancer cell targeting is further bound to the surface of the quantum dots.

The quantum dot for molecular imaging according to the present invention synthesizes cadmium-selenium particles at a high temperature to form a shell portion using zinc and sulfur, and after introducing cysteamine on the surface of the quantum dot by ligand exchange method, And antibodies for cancer targets can be bound.

Therefore, according to the second aspect of the present invention, the present invention comprises a first step of synthesizing a quantum dot consisting of a cadmium selenide core and a zinc sulfide shell; Introducing a cysteamine to the surface of the synthesized quantum dots to modify the surface of the quantum dots hydrophilically; And a third step of introducing a water-soluble monosaccharide into the hydrophilic surface-modified quantum dot.

In the present invention, the cysteamine of the second step may be introduced to the surface of the quantum dot by ligand exchange by ultrasonic treatment. In one embodiment of the present invention, the quantum dot nanoparticles are placed in a vial, dissolved in chloroform, an aqueous solution containing an excess of cysteamine is added, sonicated until the chloroform layer becomes transparent, and then the aqueous layer is collected. The reaction cysteamine was removed and then lyophilized to obtain a hydrophilic quantum dot surface-modified with cysteamine.

In the present invention, the water-soluble monosaccharide introduced in the third step may be selected from the group consisting of gluconic acid, citric acid, propionic acid, butyric acid and oleic acid. Preferably, gluconic acid can be selected. Such gluconic acid improves blood circulation time reduction by macrophages, thereby reducing blood intake by Cooper cells, and increases blood circulation time. If too much, the gluconic acid may not be able to bind antibodies or very small amounts. Therefore, preferably the gluconic acid and the hydrophilic surface-modified quantum dots are combined in a weight ratio of 1: 1 to 30.

In addition, the present invention relates to a method for producing a biocompatible molecular imaging quantum dot, characterized in that it further comprises the step of binding the antibody or peptide for cancer cell target to the surface of the hydrophilic quantum dot Gluconic acid bound.

In one embodiment of the present invention, by using sulfosuccinimidyl 6- [3 '(2-pyridyldithio) -propionamido] (SPDP) having two functional groups, SPDP in which cysteamine-quantum dot nanoparticles to which gluconic acid is bound is dispersed To the buffer solution, SPDP was dissolved in DMSO, added, and then reacted for 6 hours, washed, and then reacted for 12 hours by adding the antibody to bind the antibody.

The quantum dot produced by the present invention has a functional site where cysteamine is introduced, well dispersed in water, and a target ligand can be introduced, and gluconic acid is introduced to increase water solubility and circulation time in the blood, and to accumulate in vivo. It has the advantage of reducing and discharged in vitro. In addition, it can be applied as a fluorescent probe for molecular imaging in vivo by binding a ligand that can target a specific organ or cell.

Hereinafter, although this invention is demonstrated in detail, this invention is not limited to this.

Example 1 Preparation Method of Quantum Dot for Biocompatible Molecular Imaging

Step 1. Synthesis step of quantum dots using organic fluorescent material

 Add 1.6 g TOPO and 0.8 g HDA to a three-necked round flask, purge with nitrogen, heat to 280 ° C, and quickly add a TOP solution of 22 mg of cadmium acetate and 32 mg of selenium. After a predetermined reaction time, toluene was added to stop the reaction, diluted with toluene, precipitated using methanol, and the quantum dot nanoparticles were obtained by centrifugation and vacuum drying.

Step 2. Introduction of Cysteamine

To introduce cysteamine into the quantum dot nanoparticles prepared in step 1, 50 mg of quantum dot nanoparticles are placed in a 20 ml vial, dissolved with chloroform, and an aqueous solution containing excess cysteamine is added. After sonication until the chloroform layer becomes transparent, the aqueous layer is collected to remove unreacted cysteamine, and lyophilized to obtain hydrophilic quantum dot nanoparticles coated with cysteamine.

Step 3. Introduction of Gluconic Acid

In step 2, 1 mg of cysteamine-coated quantum dot nanoparticles are dissolved in MES buffer (pH 5.8) solution, and then 1 mg -30 mg of gluconic acid is added. 1-ethyl-3- (3-dimethylamino) -propyl) (1-ethyl-3- (3- (dimethylamino) -propyl, EDC) and N-hydroxysuccinimide (NHS) are added After the reaction for 24 hours or more, it is washed.

Step 4. Introduction of Antibodies for Cancer Targets

In order to introduce the antibody into the quantum dot nanoparticles synthesized in step 3, the antibody was first bound using sulfosuccinimidyl 6- [3 '(2-pyridyldithio) -propionamido] (SPDP). After SPDP was dissolved in DMSO and added to PBS buffer solution for SPDP in which quantum dot nanoparticles were dispersed, it was reacted for 6 hours, washed, and then reacted for 12 hours by adding 500 μg of antibody. After washing, stored in the refrigerator was used for the next experiment.

Example 2 Quantum Dot Analysis and Animal Image for Biocompatible Molecular Imaging

2-1. Transmission electron microscope

The quantum dot nanoparticles prepared in step 1 were analyzed by a transmission electron microscope (TEM, Transmittane electron microscopy, H-7650, Hitachi-Ltd, JAPAN) and are shown in FIG. 2.

As shown in FIG. 2, the prepared quantum dot nanoparticles not only have a diameter of 3-5 nm, but also have a uniform distribution.

2-2. Optical image acquisition

3 shows the results obtained through the IVIS optical signals from cysteamine-coated quantum dot nanoparticles synthesized in Example and cysteamine-coated quantum dot nanoparticles including gluconic acid. Each of the synthesized quantum dots was injected into the mouse and an optical image was obtained. Synthetic iron oxide nanoparticles were injected into the mouse tail vein, and images were taken. Organs were extracted and optical images were obtained.

As shown in FIG. 4, cysteamine-coated quantum dot nanoparticles were distributed mainly in the lungs, and mice died within 5 minutes after injection, and quantum dot nanoparticles including gluconic acid mainly prolonged blood circulation and mainly through the kidneys. It can be seen that the discharge. In addition, it can be seen that the amount released through the kidney increases with increasing amount of gluconic acid.

As described above, the present invention, by introducing a cysteamine in quantum dots and gluconic acid (gluconic acid) to solve the toxicity problem and short blood circulation time accumulated in certain organs in vivo by the cysteamine in vivo It may be used for molecular imaging, and may be useful for effectively diagnosing cancer by binding an antibody or peptide for cancer targeting.

1A is a schematic diagram showing a manufacturing process of an embodiment of the present invention;

1B shows cysteamine quantum dots coated with gluconic acid.

2 is a transmission electron microscope (TEM) photograph of the quantum dot nanoparticles of the present invention.

3 is an IVIS 'optical image of cysteamine-coated quantum dot nanoparticles and gluconic acid-cysteamine-quantum dot nanoparticles according to one embodiment of the present invention.

4 is an ex-vivo IVIS® optical image obtained by intravenously injecting cysteamine-coated quantum dot nanoparticles (a) and gluconic acid-cysteamine-quantum dot nanoparticles (bd) of an embodiment of the present invention into mice ( b) is 10 mg of gluconic acid, (c) is 20 mg of gluconic acid, and (d) is a quantum dot synthesized by treating 30 mg of gluconic acid;

Claims (7)

A quantum dot for biocompatible molecular imaging, characterized in that cysteamine and a water-soluble monosaccharide are bonded to a surface of a quantum dot made of a cadmium selenide core and a zinc sulfide shell. The quantum dot of claim 1, wherein the water-soluble monosaccharide is selected from the group consisting of gluconic acid, citric acid, propionic acid, butyric acid and oleic acid. The quantum dot for biocompatible molecular imaging according to claim 1 or 2, wherein an antibody or peptide for cancer cell targeting is further bound to the surface of the quantum dot. A first step of synthesizing a quantum dot composed of a cadmium selenide core and a zinc sulfide shell; Introducing a cysteamine to the surface of the synthesized quantum dots to modify the surface of the quantum dots hydrophilically; And And a third step of introducing a water-soluble monosaccharide into the hydrophilic surface-modified quantum dot. The method of claim 4, wherein the water-soluble monosaccharide introduced in the third step is selected from the group consisting of gluconic acid, citric acid, propionic acid, butyric acid and oleic acid. 6. The method of claim 5, wherein the gluconic acid introduced in the third step is 1 to 30 times (w / w) of hydrophilic surface-modified quantum dots. The quantum dot according to any one of claims 4 to 6, further comprising a fourth step of binding an antibody or peptide for cancer cell targeting to the surface of the quantum dot after the third step. Manufacturing method.

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