WO2012102516A2 - Nanocomposite having both magnetic properties and luminous characteristics and preparation method thereof - Google Patents

Abstract

The present invention relates to a nanocomposite having both magnetic properties and luminous characteristics by bonding cadmium selenide/zinc sulfide core shell quantum dots with iron oxide colloidal nanoclusters, and a preparation method thereof. The invention can be applied to cell separation using both FACS and MACS due to high magnetism and high luminous efficiency even in an aqueous solution.

Description

Nanocomposites Having Magnetic and Luminescent Properties Simultaneously and Methods for Manufacturing the Same

The present invention relates to a nanocomposite, and more particularly, to a nanocomposite having both magnetic properties and luminescent properties useful for cell separation.

Recently, nanoparticles are rapidly developing in the biomedical field because of their excellent optical and magnetic properties. Magnetic nanoparticles have been widely applied to biomedical fields such as magnetic resonance imaging (MRI), drug delivery systems, hyperthermia, and probe biosystems by using superparamagnetic properties that appear in small sizes.

In particular, iron oxide nanoparticles, among many kinds of magnetic nanoparticles, have been approved by the Food and Drug Administration (FDA) in terms of biocompatibility and biostability. Until now, magnetic nanoparticles have been synthesized through various synthetic methods such as hydrothermal reaction, thermal decomposition, electrospray synthesis, sol-gel synthesis, microemulsion synthesis, and sonochemical reaction. Has been developed. Accordingly, magnetic nanoparticles having desired sizes and magnetic properties can be appropriately used for various applications according to various methods.

Recently, a lot of researches are being used for biological applications by combining quantum dots, gold and silver nanoparticles with magnetic nanoparticles. There is an advantage that can be used by using the surface plasmon resonance effect of the quantum dot or the metal material such as gold and silver that can change the energy bandgap by adjusting the size.

Magnetic / optical complexes are being used for light emitting imaging, magnetic resonance imaging, magnetic separation, dark field imaging, and surface-enhanced Raman spectroscopy. In the meantime, studies using magnetic nanoparticles have often deteriorated magnetic properties when silica particles or nanoparticles having luminescent properties are combined.

However, in 2010, a study by the Yadong Yin Group succeeded in synthesizing a superparamagnetic iron oxide colloidal nanocluster wrapped with gold nanoshells with adjustable optical properties using iron oxide colloidal nanoclusters.

The iron oxide colloidal nanocluster has the advantage of having super magnetic properties and strong magnetic properties that can be controlled in size from nano to micro units. In addition, in the field of biocomplexes, the size of the complex is not easily absorbed in vivo after treatment and drug delivery in the case of micro-units, but is released in vitro, but the size of the nano-particles may be a problem because they are small enough to cross the cell wall . For this reason, micro-sized particles that are bound outside the cell during cell separation can be easily removed to obtain intact cells because they are bound to the surface even after the cells are separated, and can be used again after treatment. Nanoparticles, on the other hand, are absorbed into cells and are difficult to remove.

Currently, the separation of FACS using optical properties or MACS using magnetic properties is performed in terms of cell separation, and complementary use of FACS and MACS is necessary to increase cell separation efficiency. Therefore, the development of nanoclusters having both magnetic and optical properties that can be simultaneously applied to MACS and FACS is required for effective cell separation. In addition, magnetic-optical nanocomposites can be confirmed through MRI using magnetic properties or confocal laser scanning microscopy using optical properties, which is effective for more accurate diagnosis. In addition to diagnosis, hyperthermia therapy using magnetic particles, photodynamic therapy using optical particles, and methods of combining biomaterials on the surface can be used for selective treatment. As such, research on the Theranostic Tool, which simultaneously diagnoses and treats magnetic-optical nanocomposites, has been actively conducted in recent years and has a great impact on nanobiotechnology.

The present invention can be applied to fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) at the same time, the nanocomposite having both magnetic and luminescent properties useful for cell separation and its It is to provide a manufacturing method.

The present invention provides a nanocomposite having both magnetic properties and luminescence properties, wherein a cadmium selenide / zinc sulfide coreshell quantum dot and an iron oxide colloidal nanocluster are S-H bonded.

In the nanocomposite according to the present invention, the ratio of cadmium: selenium: zinc: sulfur of the cadmium selenide / zinc sulfide coreshell quantum dots is preferably 0.1: 0.2: 2-6: 2-4, respectively. It is judged that the luminous efficiency of the quantum dot is the highest in this composition.

In addition, in the nanocomposite according to the present invention, the bonding ratio of the cadmium selenide / zinc sulfide coreshell quantum dot and the iron oxide colloidal nanocluster is preferably 1: 20-40 by weight. This weight ratio appears when the quantum dots are uniformly coated on the iron oxide nanocluster surface.

In addition, the nanocomposite according to the present invention is a nanoparticle cluster, the size is preferably 100-500 nm. If the size of the cluster is too small, certain cells will non-selectively swallow nanoparticles inside the cell, which will prevent them from separating normal cells. Therefore, particles of about 100-500 nm size are preferred for cell separation as in the present invention. In addition, simply increasing the size of Fe ₃ O ₄ becomes a ferromagnetic material, the iron oxide particles are attached to each other, so the Fe ₃ O ₄ clusters are preferably agglomerated small particles.

Since the nanocomposite according to the present invention has both magnetic and luminescent properties, it can be used for cell separation using fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) simultaneously. have.

The present invention

1) Iron oxide (Fe ₃ O ₄ ) colloidal nanoclusters coated with polyacrylic acid (PAA) were treated with N- (3dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC) / N-hydroxysulfate. Coupling a surface of the thiol group by adding cysteamine after coupling with sodium sulfide-NHS;

2) dispersing the iron oxide colloidal nanocluster whose surface is substituted with a thiol group in a solution, and then adding a cadmium selenide / zinc sulfide (CdSe / ZnS) nanoparticle solution;

3) provides a method of manufacturing a nanocomposite having both magnetic properties and luminescent properties, characterized in that it comprises forming a nanocluster using a magnet while stirring and washing the solution.

At this time, the iron oxide (Fe ₃ O ₄ ) colloidal nanocluster solution 1a) FeCl ₃ · 6H ₂ O was added to ethylene glycol, poly (acrylic acid) and NaAc in a mixed solution to dissolve and then 200-220 ℃ Reacting after heating the temperature up to; 1b) the reaction product may be prepared by cooling to room temperature, followed by washing and centrifugation.

In addition, the cadmium selenide / zinc sulfide (CdSe / ZnS) nanoparticles solution is 2a) mixing the CdO and Zn (ac) ₂ and oleic acid and then heating, removing oxygen and moisture; 2b) 1-octadecene was added to the mixed solution, and the temperature was heated to 280-300 ° C., followed by reaction by adding a trioctylphosphine solution in which selenium powder and sulfur were dissolved, and then surfaced by adding 1-octane thiol. Passivating the; And 2c) cooling the solution to room temperature and then centrifuging the solution.

The cadmium selenide / zinc sulfide core shell quantum dots and the iron oxide colloidal nanocluster-conjugated nanocomposite according to the present invention can proceed with MACS and FACS simultaneously, thereby increasing the efficiency of cell separation. The present invention uses the iron oxide colloidal nanocluster to enhance the magnetic properties than the general nanoparticles, the optical properties are less photochemical destruction than the composite using the organic dye using the quantum dot is sensitive to absorption / light emission at a specific wavelength Made it possible. Accordingly, the nanocomposite according to the present invention has high magnetic properties and shows high luminous efficiency even in aqueous solution, and can be applied to cell separation using FACS and MACS simultaneously.

1A is a TEM image of CdSe / ZnS nanoparticles.

1B is an image showing electron diffraction (SAED) of CdSe / ZnS nanoparticles.

1C is a lattice image for CdSe / ZnS nanoparticles.

1D is a photograph in which nanocrystals are dispersed in chloroform in a vial.

FIG. 2A is an SEM image of the iron oxide nanoclusters, with cluster sizes ranging from 200-400 nm.

2B is a high resolution TEM image of the iron oxide nanoclusters.

2C is a low resolution TEM image of iron oxide nanoclusters.

FIG. 2D is a graph of iron oxide nanoclusters measured with a vibrating sample magnetometer (VSM).

2E is a graph of the XRD results of the iron oxide nanoclusters.

3A is an SEM image of a nanocomposite synthesized according to the present invention.

3B is a grating analysis result in a high resolution TEM image.

3c shows a lattice structure of CdSe / ZnS nanoparticles.

3d shows a lattice structure of iron oxide.

4A is an image of a confocal laser scanning microscope of a nanocomposite synthesized according to the present invention.

4B image is a mapping result for STEM-EDS.

Figure 4c is a result of the superconducting quantum interference device (SQUID) of the iron oxide (red dotted line) and the nanocomposite synthesized according to the present invention.

4d is a light emission photograph before (left) and after (right) the magnetization through the permanent magnet in distilled water.

Hereinafter, the present invention will be described in more detail with reference to examples and drawings.

Nanocomposites according to the present invention are characterized by having both magnetic properties and luminescent properties, and are suitable for use in the vertical separation process. More specifically, since the nanocomposite according to the present invention has both magnetic and luminescent properties, cell separation using fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) simultaneously is performed. Can be used for

Nanocomposites according to the invention are clusters of nanoparticles, the size of the clusters being about 200-400 nm. In the past, nanoparticle clusters having a size of several nm to several tens of nm have been reported, but there have been no reports of forming clusters of the size according to the present invention. In addition, when the size of the cluster is too small, certain cells non-selectively swallow the nanoparticles inside the cell, and this will not be able to separate the normal cells, the present inventors have made a large particle so that the cells do not swallow.

In addition, simply increasing the size of Fe ₃ O ₄ becomes a ferromagnetic material, the iron oxide particles are attached to each other, so Fe ₃ O ₄ was synthesized in the form of clusters of small particles.

The present invention will be described in more detail with reference to the following Examples. However, the examples are only for illustrating the present invention, and the scope of the present invention should not be construed as being limited to the examples.

Example

Cadmium oxide (CdO, 99.99%), zinc acetate (Zn (ac) ₂ , 99.99%), selenium powder (99.99%), sulfur (99.998%), iron (III) chloride hexahydrate (FeCl ₃ · 6H ₂ O, 97%), poly (acrylic acid) (PAA, Average M _w ~ 1,800), sodium acetate (NaAc, 99.0%), N- (3dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC), N -Hydroxysulfosuccinimide sodium salt (Sulfo-NHS, 98.5%), cysteamine (-95%), oleic acid (99%), 1-octadecene (90%), trioctylphosphine (TOP, 90 %), Octane thiol (98.5 +%), ethylene glycol (99.8%), toluene (99.8%), phosphate buffer solution (PBS, 1.0 M, pH 7.4) were purchased from Sigma-Aldrich, and ethanol (99.9% ) Was purchased from JT Baker and all used without further purification.

Example 1 Preparation of Cadmium Selenide / Zn Sulfide (CdSe / ZnS) Coreshell Quantum Dots

13 mg (0.1 mmol) of CdO and 732 mg (4 mmol) of Zn (ac) ₂ , 5 mL of oleic acid are placed in a 100 mL three-necked flask and heated to 150 ° C., nitrogen bubbling for 30 minutes. Oxygen and moisture were removed through. Subsequently, 15 mL of 1-octadecene is added to the mixed dispersion solution of nitrogen atmosphere, and the temperature is raised to 300 ° C. In this case, a yellowish transparent solution is formed.

Then, rapidly add 2 mL of trioctylphosphine solution containing 16 mg (0.2 mmol) of selenium powder and 96 mg (3 mmol) of sulfur into a three-necked flask and react for 8 minutes. After a while, the temperature is raised and 1-octanethiol is added to passivate the surface through strong bonding with the surface of the product.

After cooling to room temperature, add 15 mL of ethanol and centrifuge for 10 minutes at 4000 rpm. Discard the supernatant and disperse the precipitate in 10 mL of chloroform, add 30 mL of ethanol, and centrifuge at 4000 rpm for 10 minutes to perform three washes, and then disperse in 30 mL of toluene.

Example 2 Preparation of Iron Oxide (Fe ₃ O ₄ ) Colloidal Nanoclusters

Add 0.675 g (2.5 mmol) of FeCl ₃ · 6H ₂ O to 25 mL of ethylene glycol, wait until it becomes an orange clear solution, and then add 720 mg of poly (acrylic acid) and 2.16 g of NaAc in the mixed solution. After dissolving the solid reagent sufficiently by ultrasonication, the reaction mixture was placed in a 35 mL stainless steel autoclave and heated to a temperature of 220 ° C. through a furnace, followed by reaction for 4 hours. After dropping the product to room temperature, add 20 mL of distilled water and 20 mL of ethanol, and proceed with a centrifuge for 10 minutes at 4000 rpm. After leaving the precipitate and discarding the supernatant, 40 mL of distilled water was added and centrifuged at 4000 rpm for 10 minutes to remove the unreacted impurities until the supernatant became colorless. It is dispersed in a phosphate buffer solution of pH 7.4.

Example 3 Binding of Cadmium Selenide / Zn Sulfide (CdSe / ZnS) Nanoparticles to Fe ₃ O ₄ Colloidal Nanoclusters

0.2 mmol of EDC and 0.1 mmol of Sulfo-NHS were added to 3 mL of Fe ₃ O ₄ colloidal nanoclusters dispersed in PBS solution and maintained for 15 minutes in an ultrasonic state. Thereafter, 0.2 mmol of cysteamine was added to the solution, followed by stirring at room temperature for 2 hours, followed by coupling, followed by washing with ethanol and distilled water.

The iron oxide colloidal nanocluster substituted with the thiol group was dispersed in 3 mL of PBS solution, and then 3 mL of CdSe / ZnS nanoparticle solution dispersed in toluene was added to form a solution separated into two layers. (The solution of the separated layer is maintained for 24 hours with stirring. The permanent solution is used to remove the remaining CdSe / ZnS nanoparticles by alternately distilled water and toluene. Nanoclusters having both optical and magnetic properties are formed.

Experimental Example: Confirmation of luminescence and magnetic properties

According to the above embodiment, the nanocomposite was formed using both the quantum dot and the magnetic nanoclusters, the EDC / sulfo-NHS coupling process, and the S-H chemistry, which were excellent in both magnetic properties and luminescence properties. Nanoparticle quantum dots with green luminescence and high quantum efficiency are achieved through a small cadmium selenide core and zinc sulfide shell. Cadmium selenide has a significant decrease in quantum efficiency due to the characteristic of selenium oxidation in aqueous solution, but when zinc sulfide shell is formed, it increases quantum efficiency through the quantum well structure of Type-I core-shell structure and at the same time passivates the surface. Through the oxidation of selenium can also be prevented it can prevent the luminous effect is significantly reduced in the aqueous solution.

Through the above examples, quantum dots showing quantum efficiencies of 60-80% in organic solvents and 15-25% in aqueous solutions were synthesized, and high resolution transmission electron microscopy (HR-TEM) analysis was performed on nanocrystals. Was performed.

1A is a TEM image of multiple nanocrystals in isolated form. 1B shows selected area electron diffraction (SAED) at selected areas for nanocrystals with high crystallinity. 1C is a TEM image for nanocrystals. 1D is a photograph in which nanocrystals are dispersed in chloroform in a vial. The pictures on the left and right were before and after applying the 365 nm wavelength light using UV-Lamp, respectively, and the green light emission was confirmed. The magnetic colloidal nanoclusters were modified by Yadong Yin's experiment. PAA-coated water-soluble magnetic nanoclusters were synthesized to form multifunctional composite nanoparticles for biomedical use. Water-soluble nanoparticles were synthesized without substitution of ligand through synthesis using polyacrylic acid (PAA) as a surfactant. Zeta Potential value was -49 mV and showed good stability. It is easily designed when combined with quantum dots.

As shown in the SEM image of FIG. 2A, the cluster size was 200-400 nm. Each nanoparticle is 6 nm in size with superparamagnetic properties, as shown in the high resolution TEM image of FIG. 2B, and (220) lattice fringes were clearly observed. The graph of FIG. 2D is a data measured by a vibrating sample magnetometer (VSM), and shows a superparamagnetic characteristic with a saturation magnetization value of about 84 emu / g and a residual magnetization value of zero. Since the size of the magnetic nanoclusters is in micro units, it may be advantageous that the magnetic nanoclusters may be released in vitro without being easily absorbed in vivo after treatment and drug delivery. Figure 2e is the XRD results through which the synthesized results confirmed that the Fe ₃ O ₄ (Magnetite) nanoclusters.

The synthesized cadmium selenide / zinc sulfide core-shell quantum dots and iron oxide colloidal nanoclusters were used to form a nanocomposite having both magnetic and luminescent properties. EDC / sulfo-NHS coupling was performed using an acid group on the surface of the iron oxide colloidal nanocluster coated with polyacrylic acid (PAA), and cysteamine was used to form an amide bond. Iron oxide colloidal nanoclusters surface-treated with SH groups were formed.

Since the S-H group is present on the surface of the magnetic nanocluster, an attempt was made to combine the S-H chemical reaction with a cadmium selenide / zinc sulfide coreshell quantum dot dispersed in an organic solvent. Analysis was attempted to confirm the synthesis of the optical nanocomposites having the magnetic and luminescent properties bound as described above.

3A is an SEM image of a nanocomposite synthesized according to the above embodiment. In the experiment, it was confirmed that the boundary on the image was blurred when the quantum dot was not bonded to each iron oxide particle and the aggregation occurred. When quantum dots are coupled to a single nanocluster, it was confirmed that each interface is clearly shown as shown in FIG. 3a. 3b attempts to separate the combined iron oxide nanoclusters and quantum dot nanoparticles through lattice analysis in high resolution TEM images. Inverse FFT was attempted after masking the grating using a fast-fourier transform (FET) image obtained in FIG. 3B. Thus, the lattice structure shown in Fig. 3c represents the position of the quantum dots in Fig. 3b and the lattice structure of iron oxide in Fig. 3d. Since the quantum dots are coupled to the surface of the formed iron oxide colloidal nanocluster, many intact gratings are shown in FIG. 3C, while many gratings are visible between the quantum dots in FIG. 3D. In order to confirm that the quantum efficiency of the nanocomposite is lowered, a confocal laser microscope was performed.

The image shown in FIG. 4A shows the green emission of each of the composites. The scale bar shows that each nanocomposite has a size of about 400 nm and a yield of 75% or more is obtained. Confocal laser microscopy was confirmed as a result of the product separated using the permanent magnet in the washing process, so it was confirmed to have both magnetic and luminescent properties. This result is consistent with that shown in FIG. 4D. When the magnetic-optical nanocomposite dispersed in distilled water is excited at 365nm using UV-Lamp, green light is emitted throughout the solution, and when the magnetic field is applied through the permanent magnet, particles that emit green light are collected only near the magnet. Does not indicate. In the case of semiconductor quantum dots, the luminous efficiency is significantly decreased in aqueous solution, but as confirmed above, the nanocomposites invented still showed high quantum efficiency. FIG. 4b is a mapping result for STEM-EDS, in which the ratio of cadmium selenide / zinc sulfide precursor is cadmium: selenium: zinc: sulfur = 0.1: 0.2: 4: 3 It only performed. As a result, it could be confirmed that iron, zinc, and sulfur were evenly spread throughout the nanoclusters. Also, considering that the ratio of iron is higher than that of zinc and sulfur, it was found that the iron mapping result is darker than the others. Figure 4c is the result of measuring the hysteresis curve of the iron oxide (red dotted line) and the nanocomposite (black line) synthesized according to the present invention. Although the saturation magnetization value of the nanocomposite decreased compared to the iron oxide, it was confirmed that the magnetic properties were still high.

Taken together, the formation of complexes for each nanocluster was confirmed using SEM, and the formation of even complexes through lattice analysis and STEM-EDS mapping of high resolution TEM images. It was confirmed that the composite nanoclusters having both optical and magnetic properties were successfully synthesized through the confirmation of the luminescence properties maintained by confocal laser microscope and the magnetic properties maintained by the permanent magnet. After binding, it was slightly decreased but still showed high magnetic properties. Through this result, we synthesized nanocomposite under conditions that can be used by applying MACS and FACS simultaneously in cell separation.

In addition, the magnetic properties of the colloidal nanocluster were used to enhance the magnetic properties of the conventional materials, and quantum dots were used without using organic dyes. Many applications in the biomedical field are expected in the future.

Claims (8)

1. As a nanocomposite having both magnetic and luminescent properties,

   Cadmium selenide / zinc sulfide core-shell quantum dots and iron oxide colloidal nanoclusters characterized in that the S-H bonded.
2. The method of claim 1,

   The cadmium selenide / zinc sulfide core-shell quantum dots in the nanocomposite, characterized in that the ratio of cadmium: selenium: zinc: sulfur is 0.1: 0.2: 2-6: 2-4, respectively.
3. The method of claim 1,

   The combination ratio of the cadmium selenide / zinc sulfide core shell quantum dots and iron oxide colloidal nanoclusters is 1: 20-40 by weight ratio.
4. The method of claim 1,

   The nanocomposite is a nanoparticle cluster, the nanocomposite is characterized in that the size of 100-500 nm.
5. The method of claim 1,

   The nanocomposite is a nanocomposite used for cell separation using a fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) at the same time.
6. 1) Iron oxide (Fe ₃ O ₄ ) colloidal nanoclusters coated with polyacrylic acid (PAA) were treated with N- (3dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfate. Coupling the surface with a thiol group by adding cysteamine after coupling with a sodium synimide salt (Sulfo-NHS);

   2) dispersing the iron oxide colloidal nanocluster whose surface is substituted with a thiol group in a solution, and then adding a cadmium selenide / zinc sulfide (CdSe / ZnS) nanoparticle solution; And

   3) forming a nanocluster using a magnet while stirring and washing the solution; and a method of manufacturing a nanocomposite having both magnetic and luminescent properties.
7. The method of claim 6,

   The iron oxide (Fe ₃ O ₄ ) colloidal nanocluster solution,

   1a) adding FeCl ₃ · 6H ₂ O to ethylene glycol, and then dissolving poly (acrylic acid) and NaAc in a mixed solution and then heating the temperature to 200-220 ° C., followed by reaction; And

   1b) cooling the reaction product to room temperature, and then washing and centrifuging the nano-composite having both magnetic and luminescent properties.
8. The method of claim 6,

   The cadmium selenide / zinc sulfide (CdSe / ZnS) nanoparticle solution,

   2a) mixing CdO with Zn (ac) ₂ and oleic acid followed by heating and removing oxygen and moisture;

   2b) 1-octadecene was added to the mixed solution, and the temperature was heated to 280-300 ° C., followed by reaction by addition of a selenium powder and a trioctylphosphine solution in which sulfur was dissolved, and then the surface of 1-octane thiol was added thereto. Passivating the; And

   2c) a method of manufacturing a nanocomposite having both magnetic and luminescent properties, characterized in that it is prepared according to the step of cooling the solution to room temperature and centrifugation.

Images