KR101685379B1 - Method for preparing levan nano particles, and biomedical application - Google Patents

Abstract

The present invention relates to a Levan nanoparticle for breast cancer screening and a method for producing the same. The Levan nanoparticles and the hydrophobic substance-supported nanoparticle complex according to the present invention are useful for effectively targeting breast cancer by binding to glucose transporter 5, and are useful as diagnostic compositions for diagnosing diseases, diagnostic contrast agents, and drug delivery vehicles Can be usefully used. In particular, since it can target breast cancer, it can be used specifically for breast cancer. In addition, it is possible to easily carry various hydrophobic substances in levan nanoparticles by self-assembly, which can be useful not only for diagnosing diseases but also for various medical fields.

Description

Preparation of levan nanoparticles and its biomedical application {Method for preparing levan nano particles, and biomedical application}

The present invention relates to the preparation of Levan nanoparticles and their biomedical applications, and more particularly to a method for producing Levan nanoparticle complexes, compositions for diagnosing breast cancer, diagnostic contrast agents, and drug delivery vehicles.

In order to diagnose and treat cancer early, it is important to effectively target only cancer cells. Recently, studies on the diagnosis and treatment of cancer cells through nanoparticles have been actively conducted. The ways in which nanoparticles target cancer cells include passive targeting and active targeting. Passive targeting is based on the fact that cancer cells and tissues are wider than other normal tissues, so that the size of nanoparticles can be controlled to transmit drugs and the like only to cancer cells. Active targeting is a method of recognizing proteins specifically expressed in cancer cells Which is a method of diagnosing and treating cancer cells by introducing sugar chains, platamers, antibodies, etc. into nanoparticles. Thus, various sugar chains and the like are used to target cancer cells.

Levan is a polysaccharide composed of β 2 → 6 -bonded fructose. It is a type of fructose polymer. It has many functional groups at its 2,1-positions. It can give various functions. It can be used as a raw material for food, feed, cosmetics and medicine It is a biopolymer with many possibilities that can be used. Levan is a naturally occurring biopolymer produced by microorganisms and has applicability in many fields.

Levan is known to have good biocompatibility and various physiological effects. However, due to the difficulty in molecular weight control and mass production, dextran gelatin, another polysaccharide polymer, has been limited in its application to biomedical applications. Previous studies have reported the use of imaging probes in fructose monosaccharides to diagnose breast cancer (Bioconjugate Chem. 2007, 18, 628-634), but studies on the use of levan for breast cancer targeting have been under way not.

The biggest problem of chemotherapy using existing anticancer drugs was that cancer drugs killed not only cancer cells but also normal cells, causing severe damage to the human body. However, since nanoparticles are very small in size of tens to hundreds of nanometers, they can be injected into the human body, easy to recognize and absorb the cells, move along the blood vessels in the body, and are called EPR (enhanced permeation and retention) It is possible to selectively kill only the cancer cells while preventing the destruction of normal cells distributed around the cancer cells by gradually releasing the cancer drug encapsulated therein while remaining relatively long around the cancer cells. However, the passive cancer cell targeting method using the EPR effect has a limitation in accurately targeting only cancer cells. In addition, there is a limitation in that the nanoparticle material for the active targeting including the target substance such as an antibody, aptamer, Development is required.

In addition, many studies have been conducted to solubilize and stabilize many drugs and contrast agents having hydrophobic properties. However, there are disadvantages such as prior studies and complicated methods and several steps are required. Therefore, a simple method is required.

Accordingly, the present inventors have conducted studies to produce nanoparticles for targeting cancer, and a new diagnostic contrast agent and drug delivery vehicle using the nanoparticles, and as a result, have found that a Levan nanoparticle and a hydrophobic substance-bearing Levan nanoparticle complex are self- ). It was confirmed that the Levan nanoparticles and nanoparticle complexes function effectively as a drug delivery vehicle for effectively targeting breast cancer and diagnosing disease, a diagnostic contrast agent, and a drug, and completed the present invention Respectively.

The present invention provides Levan nanoparticles, Levan nanoparticle complexes, and methods for producing the same, which are applicable to mammograms.

It is another object of the present invention to provide a composition for diagnosing breast cancer, a diagnostic contrast agent, and a drug delivery vehicle comprising the Levan nanoparticle or Levan nanoparticle complex.

In order to achieve the above object, the present invention provides a Levan nanoparticle, a Levan nanoparticle composite, and a method for producing the same.

The present invention also provides a composition for diagnosing breast cancer, a diagnostic contrast agent, and a drug delivery vehicle comprising the Levan nanoparticle or Levan nanoparticle complex.

The Levan nanoparticles and the hydrophobic substance-carrying Levan nanoparticle complex according to the present invention are useful for effectively targeting breast cancer by strongly binding to the glucose transporter 5, and are useful as diagnostic compositions for diagnosing diseases, diagnostic contrast agents, drug delivery vehicles . ≪ / RTI > In particular, since it can target breast cancer, it can be used specifically for breast cancer. In addition, it is possible to easily carry various hydrophobic substances in levan nanoparticles by self-assembly, which can be useful not only for diagnosing disease but also for various medical fields.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram showing a method for producing an indocyanine green-levan nanoparticle composite according to the present invention.
FIG. 2 is a schematic view showing a method for producing a quantum dot nanoparticle-Levan nanoparticle composite according to the present invention.
FIG. 3 is a schematic view showing a method for producing a magnetite nanoparticle-Levan nanoparticle composite according to the present invention.
4 is a schematic view showing a method for producing indocyanine green-levan nanoparticle complex comprising paclitaxel according to the present invention.
FIG. 5 is a graph showing the size measurement results of the indocyanine green-levan nanoparticle composite according to the present invention. FIG.
6 is a graph showing the absorbance of the indocyanine green-levan nanoparticle complex according to the present invention.
FIG. 7 is a TEM measurement result of a quantum dot nanoparticle-Levan nanoparticle composite according to the present invention.
FIG. 8 is a graph showing the size measurement results of the quantum dot nanoparticle-Levan nanoparticle composite according to the present invention.
9 is a TEM measurement result of a magnetite nanoparticle-Levan nanoparticle composite according to the present invention.
FIG. 10 is a graph showing the size measurement results of the magnetite nanoparticle-Levan nanoparticle composite according to the present invention.
Figure 11 shows the size measurement results of the indocyanine green-levan nanoparticle complex comprising paclitaxel according to the present invention.
12 is a graph showing the results of measuring the degree of binding of Levan and GLUT5.
FIG. 13 is a diagram showing the result of imaging the cell suspension with the indocyanine green-levan nanoparticle complex according to the present invention. FIG.
Figure 14 shows the effect of GLUT5 inhibitor on the absorption of the indocyanine green-levan nanoparticle complex according to the present invention.
Figure 15 shows the results of imaging breast cancer in an animal model using the Indocyanine green-Levan nanoparticle complex according to the present invention.
16 is a graph showing the results of imaging fluorescence signals distributed in each organ using indocyanine green-levan nanoparticle complex according to the present invention.

The present invention provides Levan nanoparticles for breast cancer screening.

In addition, the present invention provides a levan nanoparticle composite for breast cancer on which a hydrophobic substance is supported.

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

Levan is a fructan, a biopolymer found in plants and some microorganisms, that contains thousands to hundreds of thousands of fructose linked together. It is an amphipathic sugar polymer that forms nanoparticles in aqueous solution and is a glucosozyme that is over- And GLUT (glucose transporter 5), and the Levan nanoparticle and nanoparticle complexes of the present invention are targeted to breast cancer due to the above characteristics.

Since the binding force of Levan nanoparticles to the glucose transporter 5 is stronger than that of fructose, breast cancer can be targeted with higher efficiency than fructose.

In the present invention, Levan having a molecular weight of tens of thousands to several tens of thousands of KDa can be used, but the present invention is not limited thereto.

The Levan nanoparticle complex of the present invention is a combination of a Levan nanoparticle and a hydrophobic substance, and the hydrophobic substance may be a hydrophobic contrast agent and / or a hydrophobic drug, but is not limited thereto.

The hydrophobic contrast agent is preferably indocyanine green, near-infrared fluorescence contrast agent, quantum dot nanoparticle, magnetic nanoparticle, contrast agent for MRI, ultrasound contrast agent or radiological contrast agent, more preferably indocyanine green (indocyanine green). The contrast agent is a substance capable of imaging cancer, and it is difficult to track and diagnose cancer for a long time due to low stability. However, the above problems can be solved by combining with Levan nanoparticles.

The hydrophobic drug may be selected from the group consisting of paclitaxel, methotrexate, doxorubicin, 5-fluorouracil, mitomycin-C, styrene maleic acid neocardinostatin Carboplatin, carmustine (BCNU), dacabazine, etoposide, or daunomycin, which are known to be useful in the treatment of cancer, But is not limited thereto.

The levan and the hydrophobic substance are bonded by self-assembly, and it is possible to easily carry various hydrophobic substances in the Levan nanoparticles without any special operation, Can be used effectively. The hydrophobic substance supported on the Levan nanoparticles is characterized by inclusion in the Levan nanoparticles.

In addition,

(a) dissolving levan in a solvent;

(b) dissolving the hydrophobic substance in a solvent; And

(c) mixing and purifying each of the solutions prepared in the step (a) and the step (b). The present invention also provides a method for producing a levan nanocomposite conjugated with a hydrophobic substance.

The production process of the Levan nanoparticle composite according to the present invention will be described in detail as follows.

The steps (a) and (b) are the steps of dissolving levan and a hydrophobic substance in a solvent to prepare respective solutions.

The hydrophobic material in step (b) may be a hydrophobic contrast agent and / or a hydrophobic drug, but is not limited thereto.

The hydrophobic contrast agent is preferably indocyanine green, near-infrared fluorescence contrast agent, Quantum Dot nanoparticle, magnetic nanoparticle, contrast agent for MRI, ultrasound contrast agent or radiological contrast agent.

The hydrophobic drug may be selected from the group consisting of paclitaxel, methotrexate, doxorubicin, 5-fluorouracil, mitomycin-C, styrene maleic acid neocardinostatin Carboplatin, carmustine (BCNU), dacabazine, etoposide, or daunomycin, which are known to be useful in the treatment of cancer, But is not limited thereto.

In the step (c), the reaction solution is reacted with each of the solutions prepared in steps (a) and (b). The reaction solution is stirred at 20 to 40 ° C for 10 to 30 hours, To obtain a Levan nanoparticle complex.

The solvent may be distilled water, phosphate buffered saline (PBS), alcohol, hexane, toluene or dimethyl sulfoxide (DMSO), but is not limited thereto and includes any solvent capable of dissolving a hydrophobic substance.

When the hydrophobic substance is a quantum dot nanoparticle or a magnetic nanoparticle,

(d) evaporating the solvent of the prepared solution; And

(d) further adding a small amount of a solvent to disperse the sample.

The present invention also provides a composition for diagnosing breast cancer comprising the Levan nanoparticle or Levan nanoparticle complex.

In addition, the present invention provides a contrast agent for breast cancer diagnosis comprising the composition for breast cancer diagnosis.

The present invention also provides a drug delivery system comprising the Levan nanoparticles or the Levan nanoparticle complex.

The drug delivery system according to the present invention can be used as a breast cancer-specific drug delivery system because it can target breast cancer.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined by the appended claims. It will be obvious to you.

Example 1 Preparation of Levan Nanoparticles and Levan Nanoparticle Compositions Containing Indocyanine Green

0.5% levan (Realbiotech) was dissolved in tertiary distilled water to prepare an aqueous solution of levan nanoparticles. Then, 4 mg of sigma-aldrich (1 mg / ml) was dissolved in tertiary distilled water, and the solution was added to an aqueous solution of levan nanoparticles dissolved first. The mixture was stirred at room temperature for 24 hours through a stirrer. After stirring, the mixture was centrifuged at 13500 rpm through a centrifugal separator to remove unreacted materials to prepare a Levan nanoparticle complex carrying indocyanine green. The precipitate was again dispersed in tertiary distilled water and refrigerated. The method for producing the indocyanine green-levan nanoparticle composite on which the indocyanine green is supported is schematically shown in Fig.

Example 2 Preparation of Levan Nanoparticle Composite Supporting Quantum-Point Nanoparticles

FIG. 2 is a schematic view showing a production method of a quantum dot nanoparticle-Levan nanoparticle composite in which quantum dot nanoparticles are supported on Levan.

As shown in FIG. 2, 50 mg of low-molecular-weight levan (Realbiotech) was dissolved in 5 ml of distilled water and then 5 mg / mL QD (Evidot 20 mg / mL in toluene Fort orange) ). After mixing for about 15 minutes using a sonicator, the solvent was evaporated using an evaporator. 1 ml of distilled water was added thereto to disperse the sample to obtain a Levan nanoparticle complex carrying the quantum dot nanoparticles.

Example 3: Preparation of magnetic nanoparticle supported Levan nanoparticle complex

FIG. 3 is a schematic view showing a manufacturing method of a magnetite nanoparticle-Levan nanoparticle composite in which magnetite nanoparticles, which are magnetic nanoparticles, are bonded to Levan.

As shown in Fig. 3, 0.25% of low molecular weight Levant (Realbiotech) was dissolved in distilled water and magnetic nanoparticles (in hexane) were added. After mixing for about 15 minutes using a sonicator, the solvent was evaporated using an evaporator. 1 ml of distilled water was added thereto to disperse the sample to obtain a magnetic nanoparticle-supported levan nanoparticle complex.

Example 4. Preparation of indocyanine green-levan nanoparticle complexes containing Taxol

Fig. 4 is a schematic view showing a method for producing nanoparticle complexes in which paclitaxel and indocyanine green, which are hydrophobic drugs, are supported on Levan.

As shown in Fig. 4, 0.5% Levan (low molecular weight, Realbiotech) was dissolved in the third distilled water. 4 mg of sigma-aldrich (1 mg / ml) was dissolved in tertiary distilled water. 5 mg of paclitaxel was dissolved in methanol, and ultrasonic waves were applied. After 24 hours, And stirred with a stirrer. Thereafter, centrifugation was performed at 13500 rpm through a centrifuge to remove unreacted materials. The precipitate was again dispersed in tertiary distilled water and refrigerated.

Experimental Example 1. Characterization of Levan nanoparticle complex

1-1. Characteristics of Levan Nanoparticles and Indocyanine Green-Levan Nanoparticle Composites

The sizes of the Levan nanoparticles and Levan nanoparticle composites prepared in Example 1 were measured with DLS (dyunamic light scattering, Otsuka, Japan) and are shown in FIG.

As shown in FIG. 5, the size of the 0.5% Levan nanoparticles was about 50 nm (left side of FIG. 5), and the size of the Levan nanoparticle complex bound with indocyanine green was 130 nm on average ).

In addition, the combination of indocyanine green and levan was confirmed by UV spectrum, which is shown in Fig. 6a.

As shown in FIG. 6A, it was confirmed that the absorbance of the Levan nanoparticle complex bound to indocyanine green appeared at 780 nm, confirming that indocyanine green was well bound.

In addition, the nanoparticles were diluted in TX-100 solution, and the absorbance was measured and compared with the result of dilution in distilled water, which is shown in FIG. 6B.

As shown in FIG. 6B, when the nanoparticles were diluted in the TX-100 solution, the absorbance shifted to a slightly longer wavelength (red shift), and it was confirmed that the absorbance was about 6 times higher than that when diluted in distilled water. This indicates that the nanoparticles loaded with the hydrophobic bond were released by the surfactant, indicating that levan and indocyanine green were bound by a hydrophobic-hydrophobic interaction.

1-2. Characteristics of Qdot nanoparticle-Levan nanoparticle complex

The shape and size of the Levan nanoparticle composite prepared in Example 2 were measured by TEM (transmission electron microscope) and DLS (dynamic light scattering). The results are shown in FIGS. 7 and 8. FIG.

As shown in FIG. 7, it was confirmed that Levan closely surrounds the QD, and as shown in FIG. 8, it was confirmed that the size of the quantum dot nanoparticle-Levan nanoparticle composite was 193 nm on average.

1-3. Characteristics of magnetic nanoparticle-Levan nanoparticle complex

The shape and size of the Levan nanoparticle composite prepared in Example 3 were measured by TEM (transmission electron microscope) and DLS (dynamic light scattering), and the results are shown in FIG. 9 and FIG.

As shown in FIG. 9, it was confirmed that Levan was well surrounded by the magnetite, and as shown in FIG. 10, it was confirmed that the size of the magnetite nanoparticle-Levan nanoparticle composite was 95 nm on average.

1-4. Characteristics of levan nanoparticle complex with paclitaxel and indocyanine green

The size of the Levan nanoparticle composite prepared in Example 4 was measured by DLS and is shown in FIG.

As shown in Fig. 11, it was confirmed that the size of the Levan nanoparticle composite was 178 nm on average.

EXPERIMENTAL EXAMPLE 2 Confirmation of Binding of Levan and Glucose Transporter 5

A 96 well ELISA plate was coated with 1% Levan to confirm binding of Levan and Glucose Transporter 5 (GLUT5; glucose transporter 5). BSA (bovine serum albumin) and fructose were also coated as control. To prevent nonspecific binding, wells coated with Levan and fructose were blocked with BSA. 0.5 μg of the recombinant GLUT5 protein was dissolved in 100 μl of the sample buffer and reacted at 37 ° C. in each well for 2 hours. This was reacted with biotin-coated anti-GLUT5 antibody for 1 hour, rinsed with washing buffer, reacted with avidin-HRP for 1 hour, treated with TMB and stopped with stop solution. The absorbance thereof was measured at 450 nm, and the results are shown in Fig.

As shown in Fig. 12, it was confirmed that the absorbance at Levan was higher than that of fructose and BSA. Indicating that recombinant GLUT5 protein and Levan bind strongly.

Experimental Example 3. Confirmation of Breast Cancer Imaging of Levan Nanoparticle Complex

(100 units / mL) and streptomycin (100 쨉 g / mL) containing 10% serum were cultured in DMEMB231 (human breast cancer cell line), KB (human nasopharyngeal cancer cell line) or A549 (human lung cancer cell line) At 37 ° C and 5% CO ₂ . HeLa (human cervical cancer cell line), SKOV3 (human ovarian cancer cell line) or NIH3T3 (mouse fibroblast) were cultured in DMEM (antibiotic added) medium containing 10% serum under the same culture conditions as above. For indocyanine green imaging, each cell was cultured on an 8 well chamber slide (1 x 10 ⁴ / well), treated with 100 μg of Indocyanine Green-Levan nanoparticle complex for 1 hour, and rinsed with PBS . The cells were then fixed with 4% paraformaldehyde and observed with a confocal microscope. The results are shown in FIG.

As shown in Fig. 13, uptake of nanoparticles in GLUT5 overexpressing MDAMB231 (human breast cancer cell line) was strongest, and indocyanine green signal was hardly visible in other cells.

In order to demonstrate that the indocyanine green-levan nanoparticle complex is absorbed through GLUT5 of breast cancer cells, the cells were treated with mercuric chloride as a GLUT5 inhibitor, and the results are shown in FIG. 14 .

As shown in Fig. 14, absorption of the indocyanine green-levan nanoparticle complex was reduced by the GLUT5 inhibitor, and fluorescence of indocyanine green was hardly observed. This indicates that the Levan nanoparticle complex enters breast cancer cells through binding to GLUT5.

Experimental Example 4. Measurement of Fluorescence Imaging in Animal Model Using Levan Nanoparticle Complex

All animal experiments were conducted in an experimental procedure approved by the Animal Care Committees of the Korea Research Institute of Bioscience and Biotechnology (KRIBB). Xenograft tumor models were derived by injecting MDAMB-231 (human breast cancer cell) cells subcutaneously in nude mice (BALB / c mice (female, 5-6 weeks old), central experimental animals, Korea). When the tumor size reached 500 m ³ , 2 mg of the indocyanine green-levan nanoparticle complex prepared in Example 1 was administered by intravenous injection. After a period of time, in vivo indocyanine green imaging was confirmed using the IVIS Lumina imaging system (Xenogen, USA), which is shown in FIG.

As shown in Fig. 15, it was confirmed that a fluorescence signal appeared in a dark area in a mouse administered with indocyanine green-levan nanoparticle complex after 24 hours. However, in the control mice, Indocyanine Green alone, no fluorescence was observed in the cancerous part, and signals were observed in the liver part. After 48 hours, fluorescence signals were observed in the cancer cells in the indocyanine green-levan nanoparticle complex-treated group, but the indocyanine green group showed indocyanine green emission and no signal was observed.

In order to confirm the dispersion of the nanoparticles in vivo, after 48 hours of administration, the mice were dissected to observe the fluorescence signal, which is shown in Fig.

As shown in Fig. 16, fluorescence signals were observed in the liver and liver of the indocyanine green-levan nanoparticle complex administered group. This indicates that the Indocyanine green-Levan nanoparticle complex is effective in imaging breast cancer.

Claims (17)

A levan nanoparticle complex comprising at least one hydrophobic substance selected from the group consisting of fluorescent dyes, Evidot nanoparticles and magnetite nanoparticles as an active ingredient. The composition for diagnosing breast cancer according to claim 1, wherein the nanoparticle complex further comprises an anticancer agent. delete The composition for diagnosing breast cancer according to claim 1, wherein the fluorescent dye is indocyanine green. 3. The method of claim 2, wherein the anticancer agent is selected from the group consisting of paclitaxel, methotrexate, doxorubicin, 5-fluorouracil, mitomycin-C, But are not limited to, styrene maleic acid neocarzinostatin (SMANCS), cisplatin, carboplatin, carmustine (BCNU), dacabazine, etoposide, Wherein the composition is at least one selected from the group consisting of daunomycin. The composition for diagnosing breast cancer according to claim 1 or 2, wherein the hydrophobic substance and the Levan nanoparticles are supported by self-assembly. The composition for diagnosing breast cancer according to claim 1 or 2, wherein the hydrophobic substance is inclusive in levan nanoparticles. The method of claim 1, wherein the Levan nanoparticle composite comprises
(a) dissolving levan in a solvent;
(b) dissolving at least one hydrophobic substance selected from the group consisting of fluorescent dyes, Evidot nanoparticles and magnetite nanoparticles in a solvent; And
(c) mixing and purifying each of the solutions prepared in step (a) and step (b). The composition for diagnosing breast cancer according to claim 8, wherein the nanoparticle complex further comprises an anticancer agent. The composition for diagnosing breast cancer according to claim 8, wherein the fluorescent dye is indocyanine green. 10. The method of claim 9, wherein the anticancer agent is selected from the group consisting of paclitaxel, methotrexate, doxorubicin, 5-fluorouracil, mitomycin-C, But are not limited to, styrene maleic acid neocarzinostatin (SMANCS), cisplatin, carboplatin, carmustine (BCNU), dacabazine, etoposide, Wherein the composition is at least one selected from the group consisting of daunomycin. The composition for diagnosing breast cancer according to claim 8, wherein the solvent is selected from the group consisting of distilled water, alcohol, hexane, toluene, dimethyl sulfoxide (DMSO), and phosphate buffered saline (PBS). 9. The composition for diagnosing breast cancer according to claim 8, wherein the mixing is performed at 20 to 40 DEG C for 10 to 30 hours. delete A levan nanoparticle complex on which at least one hydrophobic substance selected from the group consisting of fluorescent dyes, Evidot nanoparticles and magnetite nanoparticles is contained as an active ingredient. 16. The contrast agent for breast cancer diagnosis according to claim 15, wherein the nanoparticle complex further comprises an anti-cancer agent. delete

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