KR100838157B1 - Semiconductor nanoparticle-contained film and particle, and use thereof - Google Patents

Abstract

The present invention relates to a polymer film comprising a semiconductor nanoparticle, a polymer particle and a use thereof, and more particularly, the present invention relates to a polymer film comprising a semiconductor nanoparticle surface-modified hydrophobic or hydrophilic and optionally a biocompatible polymer , Polymer particles, and cell culture plates coated therewith; A cell culture substrate or cell culture plate that facilitates delivery of semiconductor nanoparticles to the cell surface or inside including the polymer film or polymer particles, and a method for delivering semiconductor nanoparticles into cells using the same; And it relates to an implant (implant) that is easy to transfer the semiconductor nanoparticles to a living tissue comprising the polymer film or polymer particles.

Description

Films and particles comprising semiconductor nanoparticles and their use {SEMICONDUCTOR NANOPARTICLE-CONTAINED FILM AND PARTICLE, AND USE THEREOF}

1 is a photograph of a polymer film including semiconductor nanoparticles of the present invention observed under a fluorescence microscope.

2A and 2B are photographs of the polymer film including the semiconductor nanoparticles of the present invention and the pure polymer film of Comparative Example 1 not including the semiconductor nanoparticles using an in vivo imaging device. 2a is a polymer film containing the semiconductor nanoparticles of the present invention, 2b is a pure polymer film.

3 is a photograph of a polymer particle including a semiconductor nanoparticle prepared in Example 2-10 of the present invention observed under a fluorescence microscope.

Figure 4 is a photograph of a breast cancer cell line EMT-6 cells cultured by the method of Example 3-1 using the polymer film containing the semiconductor nanoparticles prepared in Example 2-3 with a fluorescence microscope.

FIG. 5 is a photograph of fluorescence microscopy of NIH3T3 cells, which are fibroblasts cultured by the method of Example 3-2, using the polymer film including the semiconductor nanoparticles prepared in Example 2-3.

Figure 6a shows a result of observing the cancer cells by fluorescence microscopy after implanting the polymer film containing semiconductor nanoparticles in the embodiment 5 of the present invention to the biological region adjacent to the cancer cells, 6b is the control of no treatment To show the results.

FIG. 7A is a photograph of observing EMT-6, a breast cancer cell cultured on a plate coated with water-soluble semiconductor nanoparticles, in Example 9-1 of the present invention under a fluorescence microscope. FIG.

FIG. 7B is a photograph of observing EMT-6, a breast cancer cell, cultured on a plate not containing water-soluble semiconductor nanoparticles in Comparative Example 3-1 of the present invention under a fluorescence microscope. FIG.

FIG. 8 is a photograph taken after culturing the water-soluble semiconductor nanoparticles dispersed in Example 9-2 of the present invention and EMT-6, which is a breast cancer cell, by fluorescence microscopy.

9a is a photograph of breast cancer cells cultured on a plate coated with hydrophobic semiconductor nanoparticles in Example 9-3 of the present invention under a fluorescence microscope.

9B is a photograph of observing EMT-6, a breast cancer cell cultured on a plate not containing hydrophobic semiconductor nanoparticles, in Comparative Example 3-2 of the present invention under a fluorescence microscope.

10 is a graph showing cell viability after growing breast cancer cells on a plate coated with water-soluble semiconductor nanoparticles and a plate not containing nanoparticles in Example 10-1 of the present invention.

11 is a graph showing cell viability after culturing the water-soluble semiconductor nanoparticles dispersed in Example 10-2 and breast cancer cells together.

12 is a graph showing cell viability after culturing breast cancer cells on a plate containing no hydrophobic semiconductor nanoparticles coated plate and nanoparticles in Example 10-3 of the present invention.

FIG. 13A is a photograph showing a change in contact angle with time in a plate coated with hydrophobic semiconductor nanoparticles in Example 11 of the present invention. FIG.

FIG. 13B is a graph showing changes in contact angles on a plate coated with hydrophobic semiconductor nanoparticles and a plate not coated with nanoparticles according to Example 12 of the present invention, according to time and degree of coating of nanoparticles.

The present invention relates to a polymer film comprising a semiconductor nanoparticle, a polymer particle and a use thereof, and more particularly, the present invention relates to a polymer film comprising a semiconductor nanoparticle surface-modified hydrophobic or hydrophilic and optionally a biocompatible polymer , Polymer particles, and cell culture plates coated therewith; A cell culture substrate or cell culture plate that facilitates delivery of semiconductor nanoparticles to the cell surface or inside including the polymer film or polymer particles, and a method for delivering semiconductor nanoparticles into cells using the same; And it relates to an implant (implant) that is easy to transfer the semiconductor nanoparticles to a living tissue comprising the polymer film or polymer particles.

Semiconductor nanoparticles (semiconductor nanoparticles), also called quantum dot (quantum dot), because of its unique physical properties that emit a strong fluorescence only at a specific wavelength, has been mainly used in the manufacture of semiconductor devices such as transistors and storage media. In recent years, by utilizing the unique physical fluorescence characteristics of such semiconductor nanoparticles, the life of optical nanoparticles such as optical imaging for observing changes in the molecular level of a cell or a living body or a phenomenon occurring in real time, etc. Although research is being conducted for application to scientific research, research is mainly focused on surface modification of semiconductor nanoparticles dispersed in a colloidal phase.

Therefore, there is a need for development of appropriate technologies for application to various life science researches using the advantageous properties of semiconductor nanoparticles.

In response to such demands, the present invention aims to produce polymer films and particles containing semiconductor nanoparticles.

Still another object of the present invention is to provide a technology for delivering semiconductor nanoparticles to or into a cell surface by using a polymer film and / or polymer particles containing semiconductor nanoparticles as a substrate for cell culture.

It is still another object of the present invention to provide a technique for transferring a semiconductor film to a desired biological site by implanting a polymer film and / or a polymer particle including the semiconductor nanoparticle into a living body.

It is still another object of the present invention to provide a technique for observing a cell or a biosite by a spectroscopic method using a polymer film and / or a polymer particle including semiconductor nanoparticles.

The present invention relates to a polymer film comprising a semiconductor nanoparticle, a polymer particle and a use thereof, and more particularly, the present invention relates to a polymer film comprising a semiconductor nanoparticle surface-modified hydrophobic or hydrophilic and optionally a biocompatible polymer , Polymer particles, and cell culture plates coated therewith; A cell culture substrate or cell culture plate that facilitates delivery of semiconductor nanoparticles to the cell surface or inside including the polymer film or polymer particles, and a method for delivering semiconductor nanoparticles into cells using the same; And it relates to an implant (implant) that is easy to transfer the semiconductor nanoparticles to a living tissue comprising the polymer film or polymer particles.

First, the present invention provides a polymer film, a polymer particle or a plate coated with semiconductor nanoparticles containing the semiconductor nanoparticles.

The semiconductor nanoparticle of the present invention mainly comprises at least one member selected from the group consisting of CdSe, ZnSe, CdTe, PbSe / Te, InAs and InSe, and the core part having a diameter of about 1 to 5 nm, preferably 2 nm; It is preferred to have a core / shell structure having a total diameter of about 3 to 10 nm, including a shell portion coated with at least one selected from the group consisting of ZnS, CdS, PbS, InP, and the like for stabilization of nanoparticles.

In one embodiment of the present invention, the surface of the semiconductor nanoparticles of the core / shell structure can be used by coating with a hydrocarbon, in this case, the surface of the nanoparticles exhibit a hydrophobic property, it can be uniformly solubilized in an organic solvent. have. The hydrocarbon may be selected from the group consisting of (C4-C18). Such hydrophobic semiconductor nanoparticles may be used directly or dissolved in a nonpolar solvent such as chloroform.

Commercially available semiconductor nanoparticles are mostly coated with a hydrophilic polymer outside the core / shell structure, and semiconductor nanoparticles having a hydrophobic surface of the present invention may be obtained by forming a carbohydrate coating layer without a coating layer of the hydrophilic polymer. . In addition, since most of the biocompatible polymers are solubilized in an organic solvent, when using the semiconductor nanoparticles having a hydrophobic surface as described above, the preparation of an aqueous solution further comprising a biocompatible polymer in addition to the semiconductor nanoparticles having a hydrophobic surface It is possible.

From a semiconductor nanoparticle or a solution in which the semiconductor nanoparticle and the biocompatible polymer are dissolved, a film, a nanoparticle, a microparticle, a fiber, or a 3-D scaffold containing the semiconductor nanoparticle and the biocompatible polymer, etc. It may be used for various purposes in the field of biotechnology and medicine, such as manufacturing or coating it on a suitable substrate to prepare a plate for cell culture.

In another embodiment of the present invention, the hydrophobic surface of the semiconductor nanoparticles may be recoated with a hydrophilic polymer to prepare and use hydrophilic semiconductor nanoparticles having a diameter of about 10 nm or less. The hydrophilic polymer is preferably selected from the group consisting of polyethylene glycol, polyethyleneimine, chitosan, alginic acid, hyaluronic acid, collagen, heparin. Since the hydrophilic modified semiconductor nanoparticles are unstable when dispersed in pure water, phase separation occurs, it is preferable to store them in a buffer solution prepared to improve stability.

The biocompatible polymer that can be additionally included is a poly (L-lactide) (PLLA), a poly (DL) that can advantageously move the semiconductor nanoparticles to cells or tissues while stably supporting the semiconductor nanoparticles. -Lactide) (PDLLA), poly (glycolide) (PGA), poly-DL-lactide / glycolide copolymer (PLGA), chitosan, alginic acid, hyaluronic acid, collagen, heparin, dextran, and poly ( ε-caprolactone) is preferably selected from the group consisting of. The content ratio of the semiconductor nanoparticle and the biocompatible polymer contained in the polymer film or the polymer particle may be 1: 100000 to 1:10 (semiconductor nanoparticle weight: biocompatible polymer weight), and the ratio is 1: 10000 to 1: 100. More preferably, it is most preferable that it is 1: 1000. If the content of the semiconductor nanoparticles is less than the above range, it is difficult to observe the fluorescence due to the small amount delivered to the cells or tissues, if more than the above range, the formation of the film or particles is not easy.

The biocompatible polymer may be used in a dissolved state in a polar or nonpolar solvent. Preferably, the concentration is 0.1 to 30% (w / v), an aqueous solution, an acetic acid solution, a dimethylsulfoxide solution, a chloroform solution, and dichloromethane. It can be used in the form of a solution or acetone solution.

The polymer film including the semiconductor nanoparticles of the present invention and the biocompatible polymer may be prepared by solubilizing the semiconductor nanoparticles having a hydrophobic or hydrophilic surface in a biocompatible polymer solution, then casting the film and removing the solvent completely. , The thickness is preferably 10 nm to 2 mm. When the thickness of the polymer film of the present invention is thinner than 10 nm, the handling and operation of the film is not easy, and when the thickness of the polymer film is thicker than 2 mm, the movement of the semiconductor nanoparticles in the film is difficult. It is preferable that the thickness of a polymer film is the said range.

In addition, the polymer particles comprising the semiconductor nanoparticles and the biocompatible polymer of the present invention, after solubilizing the semiconductor nanoparticles having a hydrophobic or hydrophilic surface in the biocompatible polymer solution, such as an emulsion method, a particle maker method, ion gelation method, etc. It can be prepared by granulation using, it is preferable that the particle diameter is 100 nm to 10 mm.

The polymer film or polymer particles prepared as described above has an advantage of superior storage stability compared to semiconductor nanoparticles dispersed in a buffer solution.

The present invention also provides a polymer scaffold or polymer fiber comprising a semiconductor nanoparticle having a hydrophobic or hydrophilic surface and a biocompatible polymer. The semiconductor nanoparticles and the biocompatible polymer that can be used at this time are as described above.

The present invention also provides a cell culture substrate comprising a polymer film and / or a polymer particle containing the semiconductor nanoparticles as the cell culture substrate. In still another aspect, the present invention provides a cell culture plate in which the semiconductor nanoparticles are coated on a suitable substrate. The substrate may be selected from the group consisting of a glass substrate, a cell culture plate, a petri dish, a T-flask, a chamber slide, and the semiconductor nanoparticles are buffered when hydrophilic. If the solution is hydrophobic, it may be coated in a solution dissolved in a concentration of 5nM to 0.1mM in a non-polar solvent containing chloroform. The cell culture substrate or cell culture plate according to the present invention is characterized in that the semiconductor nanoparticles are effectively delivered to the cultured cell surface or inside. In particular, cell culture experiments on cell culture plates coated with water-soluble nanoparticles and hydrophobic nanoparticles showed that the cytotoxicity was significantly reduced when cultured on a plate coated with hydrophobic nanoparticles. Plates for cell culture have been shown to be more preferred. In addition, the hydrophobicity increased as the number of layers of the semiconductor nanoparticles coated on the plate increases. In consideration of cytotoxicity, the number of layers of the semiconductor nanoparticles coated on the plate is preferably 0.15 to 9.

In addition, the present invention is to culture the cells in a cell culture plate using the polymer film and / or polymer particles containing the semiconductor nanoparticles as a cell culture substrate or coated on the surface of the substrate, the surface of the cultured cells or It provides an effective cell delivery method of the semiconductor nanoparticles, including the step of delivering the semiconductor nanoparticles to the inside. In one embodiment of the present invention, fluorescence micrographs of cells cultured on the polymer film or polymer particles or plates comprising the semiconductor nanoparticles are shown in FIGS. 4, 5, 7a, 7b, 8, 9a and 9b. As a result of quantifying the total number of nanoparticles delivered to the cells with the number of nanoparticles contained in the polymer film, 10-30% of the nanoparticles were delivered. As such, it can be confirmed that the semiconductor nanoparticles are effectively delivered into cells cultured on the polymer film including the semiconductor nanoparticles of the present invention.

The present invention also provides an implant for a living body, which includes a polymer film and / or a polymer particle including the semiconductor nanoparticles described above, and enables effective delivery of semiconductor nanoparticles to a living tissue or cell. .

The present invention also provides a method for in vivo or ex vivo spectroscopy of a cell or a living body using the polymer film and / or polymer particles comprising the semiconductor nanoparticles.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the examples set forth below are provided to aid the understanding of the present invention, but the present invention is not limited to these examples.

[ Example ]

Example  1. Preparation of Semiconductor Nanoparticles 1

Example  1-1. Preparation of Water-Soluble Semiconductor Nanoparticles 1

Quantum Dot Corporation of the United States put a solution of Qtracker655 (non-targeted Quantum Dots, particle size approximately 20 nm) modified with polyethylene glycol in Centricon and replaced with water to remove a buffer solution and a solution of semiconductor nanoparticles Got it. The emission wavelength of the obtained semiconductor nanoparticle was 655 nm.

Example  1-2. Granularity  Preparation of Uniform Hydrophobic Semiconductor Nanoparticles

Semiconductor nanoparticles were prepared according to the Journal of the American Chemical Society, 2003, 125 , 12567-12575; Chemistry of Materials, 2003, 15 , 2854-2860. That is, CdSe core semiconductor nanoparticles having a uniform particle size of 3.8 nm were prepared, and the CdS 2 layer was coated on the nanoparticles to have a CdSe / CdS core / shell structure having a diameter of 5.2 nm, and the surface was made of hydrocarbon (C8). Hydrophobic semiconductor nanoparticles were prepared. Purified nanoparticles were dispersed in chloroform to make a stock solution (20 mg / mL). The quantum efficiency was about 25% and the emission wavelength was 607 nm.

Example  2. Preparation of Polymer Film and Polymer Particle Containing Semiconductor Nanoparticles

Example  2-1. Preparation of a polymer film containing water-soluble semiconductor nanoparticles 1

A 20% aqueous solution of 5% (w / v) chitosan (molecular weight 60,000, 2% acetic acid aqueous solution) was mixed with 0.1 ml of the aqueous solution of water-soluble semiconductor nanoparticles prepared in Example 1-1 to prepare a uniform semiconductor nanoparticle / polymer dispersion. 1 ml of the dispersion was dropped on a glass plate, pushed out to a thickness of 0.3 mm, and the solvent was evaporated at room temperature to prepare a water-soluble polymer film including semiconductor nanoparticles modified with water solubility. The emission wavelength was 565 nm as a result of observing the polymer film with a fluorescence spectrometer.

Example  2-2. Preparation of Polymer Film Containing Water-Soluble Semiconductor Nanoparticles 2

0.2 ml each of the semiconductor nanoparticles / polymer dispersion prepared in Example 2-1 was placed in a 24-well plate and dried at room temperature to prepare a water-soluble polymer film containing water-soluble semiconductor nanoparticles in each well. As a result of fluorescence microscopy, red fluorescence was observed.

Example  2-3. Preparation of Polymer Film Containing Hydrophobic Semiconductor Nanoparticles 1

1 g of PLLA (Polyscience, MW 300,000) was stirred at room temperature with a magnetic spoon and dissolved in chloroform (JT Baker) to make 20 ml solution (5% (w / v)), respectively. To the PLLA solution thus prepared, 0.05 mL of the hydrophobic semiconductor nanoparticle / chloroform solution dispersed in chloroform prepared in Example 1-3 was added and mixed well. 1 ml of the solution thus prepared was dropped on a glass plate, pushed to a thickness of 0.3 mm, and the solvent was evaporated at room temperature to prepare a polymer film containing hydrophobic semiconductor nanoparticles. When the film shows fluorescence, blue fluorescence under irradiation with blue light shows red fluorescence, and the results are shown in FIG. 1. Observation of the film with an in vivo imaging device revealed red fluorescence, which is shown in FIG. 2A.

Comparative example  1. Preparation of polymer films 1

1 g of PLLA (Polyscience, MW 300,000) was stirred at room temperature with a magnetic spoon and dissolved in chloroform (JT Baker) to make 20 ml solution (5% (w / v)), respectively. 1 ml of the polymer solution was dropped on a glass plate, pushed to a thickness of 0.3 mm, and the solvent was evaporated at room temperature to prepare a polymer film. Observation of the film with an in vivo imaging device showed no fluorescence at all, and the results are shown in FIG. 2B.

Example  2-4. Preparation of Polymer Film Containing Hydrophobic Semiconductor Nanoparticles 2

Example 2-3, except that 0.05 mL of the hydrophobic semiconductor nanoparticles / chloroform solution of Example 1-2 was added to 10 ml of 2.5% (w / v) PLLA (Polyscience, MW 50,000) chloroform solution. In the same manner, a polymer film containing hydrophobic semiconductor nanoparticles was prepared. When the film was observed with an fluorescence microscope (Olympus), it was observed that the red fluorescence.

Example  2-5. Preparation of Polymer Film Containing Hydrophobic Semiconductor Nanoparticles 3

Example 2-3, except that 0.1 mL of the hydrophobic semiconductor nanoparticles / chloroform solution of Example 1-2 was added to 10 ml of 20% (w / v) PLLA (Polyscience, MW 50,000) chloroform solution. In the same manner, a polymer film containing hydrophobic semiconductor nanoparticles was prepared. When the film was observed with an fluorescence microscope (Olympus), it was observed that the red fluorescence.

Example  2-6. Preparation of Polymer Particles Containing Water-Soluble Semiconductor Nanoparticles 1

A 20% aqueous solution of 5% (w / v) chitosan (molecular weight 60,000, 2% acetic acid aqueous solution) was mixed with 0.1 ml of the aqueous solution of water-soluble semiconductor nanoparticles prepared in Example 1-1 to prepare a uniform semiconductor nanoparticle / polymer dispersion. The dispersion was placed in a syringe and a 16 gauge needle was connected. The dispersion was pushed out of a syringe, dropped into liquid nitrogen, and lyophilized to prepare a water-soluble polymer particle containing semiconductor nanoparticles modified with water solubility. The particle size was between 2 micrometers and 3 mm.

Example  2-7. Preparation of Polymer Particles Containing Hydrophobic Semiconductor Nanoparticles 1

0.10 g of poly-DL-lactide [Poly (DL-lactide), Polyscience, USA] was dissolved in 1 ml of acetone (Junsei, JAPAN), and hydrophobic dispersed in chloroform prepared in Example 1-2 The organic phase was prepared by adding 5 mL of the semiconductor nanoparticle / chloroform solution and mixing well. 60.0 g of magnesium chloride hexahydrate (Sigma) to be used as a salting agent and 2.0 g of polyvinyl alcohol (Aldrich, USA) to be used as a stabilizer were dissolved in 100 ml of distilled water to prepare an aqueous phase. 1 ml of the organic phase and 15 ml of the aqueous phase thus prepared were mixed well using a homogenizer for 30 minutes to prepare polymer particles containing hydrophobic semiconductor nanoparticles. The size of the obtained particles was measured by Zetasizer (Malvern) to confirm that particles having an average particle size of 330 nm were produced.

Example  2-8. Preparation of Polymer Particles Containing Hydrophobic Semiconductor Nanoparticles 2

Hydrophobicity in the same manner as in Example 2-7, except that an 85/15 poly-DL-lactide / glycolide copolymer (PLGA, MW30,000, Polyscience, USA) was used instead of poly-DL-lactide Polymer particles containing semiconductor nanoparticles were prepared. The size of the obtained particles was measured by Zetasizer (Malvern), it was confirmed that the particles having a particle size of 310 nm was prepared.

Example  2-9. Preparation of Polymer Particles Containing Hydrophobic Semiconductor Nanoparticles 3

Example 2-, except that 50/50 poly-DL-lactide / glycolide (PLGA, MW30,000, Polyscience, USA) was used instead of poly-DL-lactide [Poly (DL-lactide)] A polymer particle containing hydrophobic semiconductor nanoparticles was prepared in the same manner as in 7. The particle size of the obtained particles was measured by Zetasizer (Malvern), and as a result, it was confirmed that nanoparticles having an average particle size of 310 nm were prepared.

Comparative example  2. Preparation of Polymer Particles 1

An organic phase was prepared by dissolving 0.10 g of poly-DL-lactide (Poly (DL-lactide), MW30,000, Polyscience, USA) in 1 ml of acetone (Junsei, JAPAN). 60.0 g of magnesium chloride hexahydrate (Sigma) to be used as a salting agent and 2.0 g of polyvinyl alcohol (Aldrich, USA) to be used as a stabilizer were dissolved in 100 ml of distilled water to prepare an aqueous phase. 1 ml of the organic phase and 15 ml of the aqueous phase thus prepared were mixed well for 30 minutes to prepare polymer particles. The size of the obtained particles was measured by Zetasizer (Malvern), it was confirmed that the nanoparticles having an average size of 300 to 500 nm was prepared. Observation with a fluorescence spectrometer showed no fluorescence.

Example  2-10. Preparation of Polymer Particles Containing Hydrophobic Semiconductor Nanoparticles 4

Polymer particles containing hydrophobic semiconductor nanoparticles in the same manner as in Example 2-7, except that 0.50 g of poly-DL-lactide (MW30,000, Polyscience, USA) was used. Was prepared. The result of observing the obtained particle | grains using the fluorescence microscope is shown in FIG. As shown in FIG. 3, it was confirmed that red fluorescent particles having a size of about 1.5 micrometers were prepared.

Example  2-11. Preparation of Polymer Particles Containing Hydrophobic Semiconductor Nanoparticles 5

Except for using 0.05 g of 85/15 poly-DL-lactide / glycolide copolymer (PLGA, MW30,000, Polyscience, USA) instead of poly-DL-lactide In the same manner as in Example 2-10, polymer particles containing hydrophobic semiconductor nanoparticles were prepared. Observation of the obtained particles using a fluorescence microscope confirmed that red fluorescent particles having a size of about 1.5 micrometers were prepared.

Example  2-12. Preparation of Polymer Particles Containing Hydrophobic Semiconductor Nanoparticles 6

The above example, except that 0.05 g of 50/50 poly-DL-lactide / glycolide (PLGA, MW30,000, Polyscience, USA) was used instead of poly-DL-lactide Polymer particles containing hydrophobic semiconductor nanoparticles were prepared in the same manner as in 2-10. The obtained particles were observed using a fluorescence microscope to confirm that red fluorescent particles having a size of about 1.5 micrometers were prepared.

Example  2-13. Preparation of Polymer Particles Containing Hydrophobic Semiconductor Nanoparticles 7

Polymer particles containing hydrophobic semiconductor nanoparticles were prepared in the same manner as in Example 2-7, except that poly-DL-lactide [Poly (DL-lactide), MW30,000, Polyscience, USA) was used. It was. Observation of the obtained particles using a fluorescence microscope confirmed that red fluorescent particles having a size of about 1.5 micrometers were prepared.

Example  3. Cell Culture Using Polymer Film Containing Semiconductor Nanoparticles

Example  3-1. Cell culture using polymer film containing hydrophobic semiconductor nanoparticles 1

After disinfecting the polymer film containing the semiconductor nanoparticles prepared in Example 2-3 with 70% (v / v) ethanol, 1 x 10 ⁵ breast cancer cells EMT-6 (ATCC, USA) and fibroblasts NIH3T3 (ATCC, USA) was incubated for 8 hours in an incubator (Forma) maintained at 37 ° C., 5% CO ₂ on the polymer film. After removing the cells from the film, the cells were redispersed in a LabTec (nunc, Germany) chamber and incubated for 4 hours in an incubator (Forma) maintained at 37 ° C., 5% CO ₂ . The cells were fixed and observed with a fluorescence microscope. As a result of quantitatively comparing the number of semiconductor nanoparticles delivered to the cell with the total number of semiconductor nanoparticles contained in the polymer film, it was observed that 30.8% of inorganic nanoparticles entered the cell.

Example  3-2. Cell Culture Using Polymer Film Containing Hydrophobic Semiconductor Nanoparticles 2

Except for using the polymer film containing the hydrophobic semiconductor nanoparticles prepared in Example 2-3, and incubated for 4 hours on NIH3T3 cells (ATCC, USA) on the film, Example 3-1 and Cell culture was performed in the same manner. As a result, it was observed that the semiconductor nanoparticles were efficiently delivered into the cells. The cells were fixed and observed with a fluorescence microscope. The results are shown in FIG. 5. As a result of quantifying the number of semiconductor nanoparticles delivered to the cells compared to the total number of semiconductor nanoparticles contained in the polymer film, it was observed that 13.3% of inorganic nanoparticles entered the cell.

Example  4. Cytotoxicity Measurement of Polymer Film Containing Hydrophobic Semiconductor Nanoparticles

The hydrophobic semiconductor nanoparticles / PLLA films prepared in Example 2-3 were cut into circular shapes of about 1 cm in diameter, sterilized with 70% (v / v) ethanol, and placed in 96 well-plates, respectively. x 10 ³ breast cancer cells EMT-6 (ATCC, USA) were incubated for 8, 24 and 48 hours, respectively. As a result of measuring cytotoxicity by MTT assay (J. Immunol. Methods, 1993, 157: 203-7), the cell viability was found to be 80% or more. This appeared to be almost absent.

Example  5. Biotransplantation Experiment of Polymer Film Containing Hydrophobic Semiconductor Nanoparticles

After cutting the PLLA film produced by the method of Example 2-3 to a size of about 1.5 cm in width and 1 cm in length, cancer cells were induced in a Balb / c mouse (8 weeks old, 20 g, female) induced with Lewis lung carcinoma. It was implanted into the subcutaneous tissue of the left back about 1 cm away from. After 72 hours, only the cancer tissues were removed to make 4 micron thick sections, and then the cancer tissues were observed using a fluorescence microscope. The results are shown in FIGS. 6A and 6B (control). It was observed that the semiconductor nanoparticles migrated to cancerous tissues as compared to the control group which did not receive any treatment.

Example  6. Polymers containing semiconductor nanoparticles Scaffold  And manufacturing of fibers

Example  6-1. Polymers containing hydrophobic semiconductor nanoparticles Scaffold  Produce

1 g of PLLA (Polyscience, MW 300,000) was stirred at room temperature with a magnetic spoon and dissolved in chloroform (JT Baker) to make 20 ml solution (5% (w / v)), respectively. To the thus prepared PLLA solution, 0.05 mL of the hydrophobic semiconductor nanoparticles / chloroform solution dispersed in chloroform prepared in Example 1-2 was added and mixed well. 50 ml of NaCl (Sigma, USA) was added to 1 ml of the solution, which was then placed in a 10 ml glass vial to completely evaporate chloroform. The dried film was placed in 100 ml of water and washed for 10 hours or more to completely remove NaCl. The film was lyophilized to obtain a porous scaffold.

Example  6-2. Preparation of Polymer Fibers Containing Hydrophobic Semiconductor Nanoparticles

1 g of 50/50 poly-DL-lactide / glycolide (PLGA, MW30,000, Polyscience, USA) was stirred in a chloroform (JT Baker) at room temperature using a magnetic straw to make 20 ml solution each (5 % (w / v)). To the thus prepared PLLA solution, 0.05 mL of the hydrophobic semiconductor nanoparticles / chloroform solution dispersed in chloroform prepared in Example 1-2 was added and mixed well. The mixed solution was poured onto a glass plate and then spun to prepare a fiber. The thickness of the fiber produced was about 50 mm.

Example  7. Preparation of Semiconductor Nanoparticles 2

Example  7-1. Preparation of Water-Soluble Semiconductor Nanoparticles

A solution of Qdot605 ITK (particle size about 20 nm, 2 μM), whose surface of Quantum Dot Corporation was modified with streptavidin, was placed in centricon and replaced with water to obtain an aqueous solution of semiconductor nanoparticles in which the buffer solution was removed. The emission wavelength of the obtained semiconductor nanoparticle was 605 nm.

Example  7-2. Granularity  Preparation of Uniform Hydrophobic Semiconductor Nanoparticles

Semiconductor nanoparticles were prepared according to the Journal of the American Chemical Society, 2003, 125 , 12567-12575; Chemistry of Materials, 2003, 15 , 2854-2860. That is, CdSe core semiconductor nanoparticles having a uniform particle size of 3.8 nm were prepared, and the CdS 2 layer was coated on the nanoparticles to have a CdSe / CdS core / shell structure having a diameter of 5.4 nm and the surface was made of hydrocarbon (C8). Hydrophobic semiconductor nanoparticles were prepared. The nanoparticles thus prepared were purified and dispersed in chloroform to obtain a stock solution (20 mg / mL). The quantum efficiency was about 22% and the emission wavelength was 605 nm.

Example  8. Manufacture of Plates coated with Semiconductor Nanoparticles

Example  8-1. Manufacture of plate coated with water-soluble semiconductor nanoparticles

1.25, 5, 12.5, and 25 μl of the aqueous solution of aqueous semiconductor nanoparticles prepared in Example 7-1 were mixed with sterilized tertiary distilled water using a syringe filter, respectively, and diluted to a final volume of 100 μl. 100 μl of the diluted aqueous nanoparticle solution was transferred to 24-well cell culture plates, and the well surface was evenly coated with the aqueous solution, followed by vacuum drying for 24 hours. As a result of observing the plate under a fluorescence microscope, it was observed that red fluorescence appeared by nanoparticles. On the other hand, when the experiment with distilled water alone, no fluorescence was observed.

Example  8-2. Manufacture of plates coated with hydrophobic semiconductor nanoparticles

10, 25, 50, 100 and 200 μl of the hydrophobic semiconductor nanoparticles prepared in Example 7-2 were taken and placed in 1.5 ml tubes, respectively, and chloroform (JT Baker) was mixed and diluted to a final volume of 200 μl. . The diluted hydrophobic semiconductor nanoparticle solution was dropped on a glass plate, spread evenly, and dried at room temperature for one day. The plate produced above was observed with a fluorescence spectrometer, and the emission wavelength was 605 nm.

Example  9. Cell culture using plate coated with semiconductor nanoparticles

Example  9-1. Cell culture using plate coated with water-soluble semiconductor nanoparticles

The plate prepared in Example 8-1 was sterilized with 70% (v / v) ethanol, and then washed twice with PBS. 2 x 10 ⁴ breast cancer cells EMT-6 (ATCC CRL-2755, USA) were put thereon so that the final concentrations were 5, 20, 50 and 100 nM, respectively. Then incubated for 16 hours in an incubator (Forma) maintained at 37 ° C., 5% CO ₂ . After removing the cells from the plate, the cells were dropped on the glass plate treated with poly-L-lysine and incubated in the incubator for 4 hours. The cells were fixed and observed under fluorescence microscopy. As can be seen in Figure 7a, the delivery of the semiconductor nanoparticles into the cell can be observed.

Example  9-2. Cell culture of dispersed water soluble semiconductor nanoparticles

1.25, 5, 12.5 and 25 μl of the water-soluble semiconductor nanoparticles prepared in Example 7-1 were taken and placed in a 1.5 ml tube, respectively, and the mixture was diluted twice so that the final volume was 100 μl. 2 x 10 ⁴ breast cancer cells EMT-6 (ATCC CRL-2755, USA) were added together with the diluted semiconductor nanoparticle solution to a final concentration of 5, 20, 50 and 100 nM, respectively, and then placed in a 24-well plate. Well dispersed. Then incubated for 16 hours in an incubator (Forma) maintained at 37 ° C., 5% CO ₂ . Then, after removing the cells, the cells were dropped on a glass plate treated with poly-L-lysine and incubated for 4 hours in an incubator. The cells were fixed and observed under fluorescence microscopy. As can be seen in Figure 8, it can be observed that the semiconductor nanoparticles are delivered into the cell.

Example  9-3. Cell culture using plates coated with hydrophobic semiconductor nanoparticles

The plate prepared in Example 8-2 was sterilized with 70% (v / v) ethanol and then washed twice with PBS buffer. Drop 1 x 10 ⁵ breast cancer cells EMT-6 (ATCC CRL-2755, USA) on top of them to a final concentration of 29, 72.5, 145, 290 and 580 nM, respectively, and then at 37 ° C., 5% CO ₂ . Incubated for 16 hours in a maintained incubator (Forma). After removing the cells from the plate, the cells were dropped on a glass plate treated with poly-L-lysine and incubated for 4 hours in an incubator. The cells were fixed and observed under a fluorescence microscope. The results are shown in FIG. 9A. As can be seen in Figure 9a, it can be observed that the semiconductor nanoparticles are efficiently delivered into the cell.

Comparative example  3. Cell Culture of Plates Not Containing Semiconductor Nanoparticles

Comparative example  3-1. Plate Preparation Without Hydrophobic Semiconductor Nanoparticles

200 μl of chloroform was taken out, dropped on a glass plate, flattened, and dried at room temperature for one day. The plate was observed with a fluorescence spectrometer to show no fluorescence at all.

Comparative example  3-2. Cell culture using plates containing no water soluble semiconductor nanoparticles

The plate prepared in Comparative Example 3-1 was sterilized with 70% (v / v) ethanol and washed twice with buffer. On top of that, 2 x 10 ⁴ breast cancer cells, EMT-6 (ATCC CRL-2755, USA), were incubated for 16 hours in an incubator. After removing the cells to the plate, the cells were dropped on a glass plate treated with poly-L-lysine and incubated for 4 hours in an incubator. The cells were fixed and observed under a fluorescence microscope. The results are shown in FIG. 7B. As can be seen in FIG. 7B, no cells showing fluorescence are observed.

Comparative example  3-3. Cell Culture Using Plates That Do Not Contain Hydrophobic Semiconductor Nanoparticles

Experiments were carried out in the same manner as in Comparative Example 3-2 except that 1 x 10 ⁵ breast cancer cells EMT-6 (ATCC CRL-2755, USA) were cultured on the plate prepared in Comparative Example 3-1. The results were observed with a fluorescence microscope. The cells were fixed and observed under a fluorescence microscope. The results are shown in FIG. 9B. As can be seen in FIG. 9B, no fluorescence cells are observed.

Example  10. Cytotoxicity Measurement of Plates Coated with Semiconductor Nanoparticles

Example  10-1. Cytotoxicity Measurements Using Plates Coated with Water-Soluble Semiconductor Nanoparticles

After incubating the cells for 16 hours on the plate coated with the water-soluble semiconductor nanoparticles in the same manner as in Example 9-1, MTT assay (Immunol, Methods, 1993, 157: 203-7) was performed to measure cytotoxicity. The results are shown in FIG. 10. As can be seen in Figure 10, when the concentration of the nanoparticles is 5 nM there is little cytotoxicity, but at higher concentrations it was shown that the cell survival rate is 65 to 45%.

Example  10-2. Cytotoxicity Measurement of Dispersed Water-Soluble Semiconductor Nanoparticles

After culturing the cells and the semiconductor nanoparticles dispersed in the aqueous solution at the same time in the same manner as in Example 9-2, and measuring the number of living cells using trypan blue (Gibco) to stain the membrane of the cell The results are shown in FIG. As can be seen in Figure 11, when the semiconductor nanoparticles are included, the cell survival rate is about 70-10%, it can be seen that the toxicity is relatively severe, the cell survival rate is significantly decreased as the concentration is increased. appear.

Example  10-3. Cytotoxicity Measurements Using Plates Coated with Hydrophobic Semiconductor Nanoparticles

After culturing the cells for 16 hours on a plate coated with hydrophobic semiconductor nanoparticles in the same manner as in Example 9-3, the number of living cells was measured using trypan blue (Gibco), which stains the cell membrane. The results are shown in FIG. As can be seen in Figure 12, when the semiconductor nanoparticles are included, the cell viability was found that most of the cells are alive until the concentration of the semiconductor nanoparticles 145nM, at a concentration higher than that of about 80% As a result, the plate coated with the semiconductor nanoparticles of the present invention was shown to have little cytotoxicity.

Example  11. Measurement of Contact Angles on Plate Surfaces Coated with Hydrophobic Semiconductor Nanoparticles

The number of layers of the semiconductor nanoparticles coated on the plate prepared in Example 8-2 was calculated to be 0.38, 0.96, 1.92, 3.84. 10 μl of tertiary distilled water was dropped on the glass plate coated with the nanoparticles, and the contact angles after 0, 1, 5, and 30 minutes were measured using an OPTEM camera, and the results are shown in FIGS. 13A and 13B. As can be seen in FIGS. 13A and 13B, it was observed that as the number of layers of nanoparticles increased, the contact angle increased, which means that the hydrophobicity increased.

The polymer film, the polymer particle, the polymer scaffold, and the polymer fiber including the semiconductor nanoparticles of the present invention may be used for various cell experiments and disease diagnosis, and may be used as a delivery system in combination with a bioavailable material or drug. It can be advantageously applied to various medicine and biotechnology fields.

Claims (27)

Polymer film containing a semiconductor nanoparticle of the core / shell structure, The semiconductor nanoparticle is a surface having a hydrophobic surface is coated with a hydrocarbon selected from the group consisting of 4 to 18 carbon atoms, Polymer film. The method of claim 1, wherein the semiconductor nanoparticles are one or more selected from the group consisting of CdSe, ZnSe, CdTe, PbSe / Te, InAs, and InSe, the core portion having a diameter of 1 to 5 nm, and ZnS, CdS A polymer film having a core / shell structure having a total diameter of about 3 to 10 nm, comprising a shell portion coated with at least one selected from the group consisting of PbS and InP. delete delete delete The poly (L-lactide) (PLLA), poly (DL-lactide) (PDLLA), poly (glycolide) (PGA), poly-DL-lactide / glycolide copolymers (PLGA) ), Chitosan, alginic acid, hyaluronic acid, collagen, heparin, dextran, and a polymer film further comprising at least one biocompatible polymer selected from the group consisting of poly (ε-caprolactone). The polymer film according to claim 6, wherein the content ratio of the semiconductor nanoparticle and the biocompatible polymer is 1: 100000 to 1:10 (semiconductor nanoparticle weight: biocompatible polymer weight). A polymer particle containing a semiconductor nanoparticle of the core / shell structure, The semiconductor nanoparticle is a surface having a hydrophobic surface is coated with a hydrocarbon selected from the group consisting of 4 to 18 carbon atoms, Polymer particles. The method of claim 8, wherein the semiconductor nanoparticles are at least one selected from the group consisting of CdSe, ZnSe, CdTe, PbSe / Te, InAs, and InSe, the core portion having a diameter of 1 to 5 nm, and ZnS, CdS A polymer particle having a core / shell structure having a total diameter of about 3 to 10 nm, including a shell portion coated with at least one selected from the group consisting of PbS and InP. delete delete delete The poly (L-lactide) (PLLA), poly (DL-lactide) (PDLLA), poly (glycolide) (PGA), poly-DL-lactide / glycolide copolymers (PLGA) ), A polymer particle further comprising at least one biocompatible polymer selected from the group consisting of chitosan, alginic acid, hyaluronic acid, collagen, heparin, dextran, and poly (ε-caprolactone). The polymer particle according to claim 13, wherein the content ratio of the semiconductor nanoparticle and the biocompatible polymer is 1: 100000 to 1:10 (semiconductor nanoparticle weight: biocompatible polymer weight). A substrate for cell culture, comprising: a polymer film comprising the semiconductor nanoparticles of any one of claims 1, 2, 6, and 7, wherein: 8, 9, 13, and 14 It includes a polymer particle or any one comprising the semiconductor nanoparticles of any one, The semiconductor nanoparticle is a surface having a hydrophobic surface is coated with a hydrocarbon selected from the group consisting of 4 to 18 carbon atoms, The substrate for cell culture, characterized in that the semiconductor nanoparticles are effectively delivered to or inside the cultured cell surface. A plate for cell culture in which semiconductor nanoparticles having a core / shell structure are coated on a substrate. The semiconductor nanoparticle is a surface having a hydrophobic surface is coated with a hydrocarbon selected from the group consisting of 4 to 18 carbon atoms, Plate for cell culture. 17. The method of claim 16, wherein the semiconductor nanoparticles are at least one selected from the group consisting of CdSe, ZnSe, CdTe, PbSe / Te, InAs and InSe, the core portion having a diameter of 1 to 5 nm, ZnS, CdS A cell culture plate having a core / shell structure having a total diameter of about 3 to 10 nm, comprising a shell portion coated with at least one selected from the group consisting of PbS and InP. delete delete delete The method of culturing cells using the cell culture substrate of claim 15 to deliver the semiconductor nanoparticles to the surface or inside of the cultured cells, the method of effective cell delivery of semiconductor nanoparticles. A polymer film comprising the semiconductor nanoparticles of any one of claims 1, 2, 6 and 7, and the semiconductor nanoparticles of any one of claims 8, 9, 13 and 14. Including polymer particles or all of them, The semiconductor nanoparticle is a surface having a hydrophobic surface is coated with a hydrocarbon selected from the group consisting of 4 to 18 carbon atoms, An implant for living transplantation, capable of effective delivery of semiconductor nanoparticles to living tissue or cells. delete A polymer scaffold or polymer fiber whose surface is coated with a hydrocarbon selected from the group consisting of hydrocarbons having 4 to 18 carbon atoms and which has a core / shell structure of semiconductor nanoparticles. 25. The method of claim 24, wherein the semiconductor nanoparticles are at least one selected from the group consisting of CdSe, ZnSe, CdTe, PbSe / Te, InAs, and InSe, the core portion having a diameter of 1 to 5 nm, ZnS, CdS A polymer scaffold or polymer fiber, having a core / shell structure with a total diameter of about 3 to 10 nm, comprising a shell portion coated with at least one selected from the group consisting of PbS and InP. The poly (L-lactide) (PLLA), poly (DL-lactide) (PDLLA), poly (glycolide) (PGA), poly-DL-lactide / glycolide copolymers (PLGA) ), Chitosan, alginic acid, hyaluronic acid, collagen, heparin, dextran, and a polymer scaffold or polymer fiber further comprising at least one biocompatible polymer selected from the group consisting of poly (ε-caprolactone). 18. An effective cell delivery method of semiconductor nanoparticles, characterized in that the cells are cultured using the cell culture plate of claim 16 or 17 to deliver the semiconductor nanoparticles to the surface or inside of the cultured cells.

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