KR20110096641A - A composition comprising carbon nano-materials for inhibiting proliferation of tumor cell - Google Patents

Abstract

The present invention relates to a composition for inhibiting cancer cell proliferation comprising carbon nanomaterial as an active ingredient. More specifically, the carbon nanomaterial of the present invention relates to a composition capable of specifically inhibiting proliferation of brain tumor cells without affecting the neuronal cells that do not proliferate after differentiation. By inhibiting the proliferation and migration of cancer cells, it is possible to prevent cancer cell metastasis, and can be used in combination with other anticancer agents to prevent and treat cancer diseases.

Description

A composition comprising carbon nano-materials for inhibiting proliferation of tumor cell}

The present invention relates to a composition for inhibiting cancer cell proliferation comprising carbon nanomaterial as an active ingredient. More specifically, in the composition that can be used as a carbon nanotube having a diameter of 10 to 50nm as an active ingredient, and specifically inhibit the proliferation of brain tumor cells without affecting the neuronal cells that do not proliferate after differentiation It is about.

Carbon nanotubes have been studied since 1991 by Dr. Iijima of Meijo University, Japan, who was working with electron microscopes. Carbon nanotubes have a structure in which a graphite surface is rolled round, and has a large anisotropy, various structures such as a single wall, a multi-wall, a bundle, and a diameter of 1 to 20 nm is typical.

Graphite has a strong and flat hexagonal plate-like structure due to its unique bond arrangement. The top and bottom of the film are filled with free electrons, and the electrons move in parallel with the film in discrete states. Since the graphite layer is wound in a spiral to form carbon nanotubes, the edges are bonded at different points and the electrical properties of the nanotubes are a function of structure and diameter. B46, 1804 (1992) and Phys. Rev. Lett., 68, 1579 (1992). In other words, it has been proved that the electrical properties of the same material are expressed from insulators to semiconductors and metallics due to differences in structure and diameter.

As a method of synthesizing carbon nanotubes, an electric discharge method, a thermal decomposition method, a laser deposition method, a plasma chemical vapor deposition method, a thermochemical vapor deposition method and an electrolysis method are known. Since carbon nanotubes exist mostly in the form of bundles in the synthesis step, it is important to separate and disperse carbon nanotubes one by one in order to fully utilize the excellent mechanical and electrical properties of the carbon nanotubes. In addition, since carbon nanotubes exist as a mixture of carbon nanotubes having metallic and semiconducting properties, it is preferable to use the carbon nanotubes separately according to the use. For example, in the case of carbon nanotubes used for the electrode, it is preferable to use only metallic carbon nanotubes in order to have high conductivity, and in the case of the semiconductor layer of the transistor, it is preferable to use only semiconducting carbon nanotubes.

Carbon nanotubes are also being investigated for cancer cell death. Recently, a new cancer treatment method that destroys cancer cells with high heat generated by injecting carbon nanotubes into the body of cancer patients and irradiating infrared rays has proved effective in animal experiments.

Carbon nanotubes, which are hollow cylinders of pure carbon, are known to release heat when exposed to radio frequencies. When the nanotubes are placed in tumors, these thermal effects destroy cancer cells, and these radio waves are reported to pass harmlessly through the body.

Although carbon nanotubes are used to coat carbon nanotubes with anticancer substances that will react with cancer cells, and have carbon nanotubes to have specific targets for cancer cells, vehicle or immune effects (cytokine and inflammation) of drug delivery of anticancer drugs have been progressing. Studies on cancer cell death and enhancement inhibition on the nanotubes themselves are insufficient.

The present inventors have confirmed that the carbon nanotubes themselves act as a significant toxicity to the brain tumor cells that continue to proliferate without causing toxicity to brain cells that do not proliferate because they have already differentiated and inhibit the proliferation. It was completed.

Accordingly, an object of the present invention is to provide a composition for inhibiting cancer cell proliferation using the carbon nanomaterial as an active ingredient.

The present invention provides a composition for inhibiting cancer cell proliferation using the carbon nanomaterial as an active ingredient.

Hereinafter, the present invention will be described in detail.

The carbon nanomaterial of the present invention may be carbon nanotubes or nanofibers.

Preferably, it is a carbon nanotube.

More preferably, they are multiwalled carbon nanotubes (mwCNT).

Since carbon nanotubes exist mostly in the form of bundles in the synthesis step, the carbon nanotube ropes or bundles in the entangled state are cut to a size suitable for complexing with the polymer and separated into individual carbon nanotube levels as much as possible. It is essential to develop a technology that realizes stable nanodispersion state in the polymer matrix. Overcoming phase separation, agglomeration, low dispersibility and adhesion in the polymer matrix to be combined with carbon nanotubes is the most important requirement in nanocomposites.

The carbon nanotubes should be dispersed in an aqueous solution or an organic solvent, and since the hydrophobicity (less affinity with water) of carbon nanotubes and the mutual attraction between molecules are very high, the choice of dispersant should be considered important.

The dispersant is a surfactant, which is composed of a head part and a tail part, and the head part must have affinity for the surface of the dispersoid, which is a material to be dispersed, and the tail part is a solvent to be dispersed, that is, a dispersion medium. A good dispersant is one that must be compatible with and that can act as a barrier to collisions between particles.

Dispersants for carbon nanotubes include sodium dodecyl benzen sulfonate (NaDDBS), sodium dodecyl sulfonate, TX-100, and polyvinyl pyrrolidone as aqueous dispersants. And the like can be used.

Preferably, the nutrient protein of the cell can be used as a dispersing agent of carbon nanotubes.

In one embodiment of the present invention was attempted to disperse using a serum medium (including serum media, FBS), which is a nutrient protein of the cell rather than a general surface chemical treatment to the nanomaterial. Serum media were run four times for 30 minutes each at 3 seconds on, 6 seconds off at 24% output of up to 500 watt sonication machines using tip sonication, and also by heat protein. In order to prevent their denaturation, all samples were taken in ice.

Dispersed CNTs are weaker than serum media (w / o FBS) after a certain period of time, and thus effective in dispersing nanomaterials.They do not have chemical surface coatings (COOH, NH2, OH). There is an advantage to study the nanotoxicity of carbon materials.

Preferred multi-walled carbon nanotubes of the present invention have a diameter of 10 nm to 50 nm. More preferably 30 nm.

In one embodiment of the present invention, the smaller the diameter of the carbon nanomaterial was confirmed that the more proliferation of brain tumor cells.

In addition, one embodiment of the present invention used four kinds of carbon nanomaterials. Carbon nanomaterials used are shown in the table below.

CMMs mwCNT phCNF hbCNF hlCNF diameter 30 nm 150 nm 250 nm 250 nm  Hydrophilicity degree Partial hydrophilicity Partial hydrophilicity Hydrophobic Partial hydrophilicity

Each of the nanomaterials was treated with neuronal tumor cells (U373MG) by concentration and time to perform MTT analysis. It was confirmed that mwCNT had the strongest cytotoxicity according to the concentration. In addition, it was found that hlCNF having a surface hydrocarbon (hbCNF) removed (heat treatment: reaction for 4 hours at 400 ° C.) was also toxic.

As a result, it was confirmed that the toxicity of carbon itself affects cell division, and the smaller the size and the more exposed the carbon to the surface, the better the degree of dispersion.

After incubating the U373MG cell line with a culture solution containing each nanomaterial, TUNEL analysis was performed to examine the correlation between carbon nanomaterial tumor cell apoptosis. As a result, no apoptosis was observed.

Caspase-3 was not detected in all groups when cutout caspase-3, one of the major substrates of apoptosis, was examined by Western blot.

On the other hand, in one embodiment of the present invention, the U373MG cells treated with carbon nanomaterials and examined cell migration and division, it was confirmed that all the carbon nanomaterials used in the experiments affected the cell migration and division.

In addition, the hippocampal stem cell line (HT22) before neuronal differentiation had an effect on cell migration and division.

However, the carbon nanomaterials used in the embodiment of the present invention did not show any toxicity in the neurons that did not divide anymore and completed differentiation.

In conclusion, the carbon nanomaterials of the present invention, in particular, mwCNT, phCNF, hbCNF, hlCNF do not cause toxicity to brain neurons that do not proliferate already after differentiation, and inhibit cell division and proliferation in the continuously growing brain tumor cells. I could confirm it.

Therefore, on the basis of the above fact, the present invention provides a composition for inhibiting cancer cell enhancement containing carbon nanomaterial as an active ingredient.

In another aspect, the present invention provides a pharmaceutical composition for inhibiting cancer cell enhancement containing a carbon nanomaterial as an active ingredient.

The composition of the present invention can inhibit cancer cell enhancement and migration specifically to cancer cells that are proliferating.

The composition may consist of an effective amount of the carbon nanomaterials of the present invention and pharmaceutically acceptable diluents, preservatives, solubilizers, other adjuvants and / or carriers, and may be formulated for use in a pharmaceutical unit dosage form.

The composition of the pharmaceutical unit dosage form of the present invention may be formulated according to a conventional method.

The composition of the present invention may be used in the form of an aqueous injection solution, a non-aqueous injection solution, an emulsion injection solution, a suspension injection solution or an injectable (freeze-dried) powder to be dissolved in sterile injection water when used. Various formulations, such as for other injections and parenteral administration, can be prepared according to techniques described or commonly used in books well known in the art.

Additives that may be included in the composition may be any base commonly used in injections. For example, the base may be distilled water, sodium chloride solution, mixtures of sodium chloride and mineral salts or the like, solutions of mannitol, lactose, dextran, glucose, etc., amino acid solutions such as glycine, arginine, organic acid or salt solutions, and glucose solutions. Mixing and similar solutions. Injectables may also be prepared by conventional methods such as osmotic pressure regulators, pH regulators, antiseptics such as methylhydroxybenzoate or propylhydroxybenzoate, vegetable oils such as sesame oil or soybean oil, or surfactants such as lecithin or nonionic surfactants. It can be prepared into a solution, a suspension or a colloidal solution, and the like in addition to.

In addition, the above "effective amount" refers to an amount which shows a prophylactic or therapeutic effect when administered to a patient. The dosage of the composition according to the present invention may be appropriately selected depending on the route of administration, subject of administration, age, sex, weight, individual difference and disease state. The selected effective amount can be administered once a day or divided several times a day.

Preferably, the composition of the present invention may vary the content of the active ingredient according to the degree of disease.

The composition of the present invention can be administered topically to the cancer cell site in the form of an injection prepared by the above method. It may also be used in combination with other anticancer agents to inhibit cancer cell division, enhancement and metastasis.

An effective amount of the carbon nano material included as an active ingredient in the composition of the present invention is not limited. Preferably, it may be comprised in an amount of 0.001 to 10 μg / ml.

The cancer cells of the present invention may be any kind of cancer, but is not limited to any kind.

Preferably, the brain tumor cells continue to proliferate.

According to the present invention, carbon nanotubes can be used to inhibit the promotion of cancer cells and the movement of cells, thereby preventing the exacerbation of cancer diseases. It can also be used in conjunction with other anticancer drugs, ultimately preventing and treating cancer, making a significant contribution to the anticancer market.

Figure 1 is a graph of the cell adsorption rate was performed by MTT assay to determine how much the carbon nanomaterials affect the cell adsorption in U373MG cells.
Figure 2a shows the growth test results obtained by treating the nano-materials of mwCNT, phCNF, hbCNF to the neuronal tumor cells (U373MG) by concentration and time.
Figure 2b shows the cytotoxicity test results of carbon nanomaterials, including hlCNF.
Figure 3 shows the results of performing the TUNEL assay by treating each nanomaterial to the U373MG cell line and Western blot to determine whether caspase-3 is detected.
Figure 4a shows the result of confirming the movement force of the cells in each condition for 7 hours. Blue indicates the initial starting point of the cell and red indicates the last position.
4B schematically illustrates the diagram of FIG. 4A. The total travel distance and speed under each condition.
Figure 4c shows the degree of division of cells in each condition.
Figure 5 shows the results of MTT assay by treating each carbon nanomaterial to HT22 cells before differentiation into neurons.
Figure 6 shows the results of performing the MTT assay by treating carbon nanomaterials to neurons that do not divide anymore, which differentiated HT22 cells.

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 Experimental Materials

Carbon nanomaterials used in the present invention were as shown in Table 2 below.

CMMs mwCNT phCNF hbCNF hlCNF diameter 30 nm 150 nm 250 nm 250 nm  Hydrophilicity degree Partial hydrophilicity Partial hydrophilicity Hydrophobic Partial hydrophilicity

The carbon nanomaterial mwCNT (SES, TX, USA), phCNF (PS 24, Pyrograff, USA), hbCNF and hlCNF (AG-1, Pyrograff, USA), astrocyte sarcoma (U373MG: glioma, ATCC) , USA), brain neurons (HT22: a type of hippocampal stem cells, Prof. c. Behl, University of Mainz) were used in the experiment.

In order to examine the effect of the hydrophilicity of the carbon nanomaterials on the cell was added to the carbon nanomaterials to increase the hydrophilicity through heat treatment (hlCNF).

Tip sonication of serum media (including FBS) to disperse carbon nanotubes (CNTs) with different diameters of 20 nm mwCNTs, 60-150 nm and 100-300 nm carbon nanofibers (CNFs) ) Was dispersed. 24% power up to 500 watt sonication machine was performed four times for 30 minutes each on 3 seconds on, 6 seconds off, and all samples were iced to prevent denaturation of proteins due to heat. It was carried out in a dipped state. CNTs dispersed in various proteins were weaker than serum-free media (w / o FBS) after a certain period of time, and thus were effective in dispersing nanomaterials. Nanotoxicity of pure carbon materials could be studied without COOH, NH2, OH).

Experimental Example 1 Influence of Carbon Nanomaterials on Cell Adsorption

U373MG cells were suspended in serum medium and placed in a 35 mm culture dish with 200000 cells each. Adsorbed to the floor within 3 hours by the characteristics of the cells adsorbed on the floor.

In order to investigate the effect on the cell adsorption of nanomaterials, the adsorption rate was examined by performing MTT assay 1 hour and 3 hours after cell culture.

MTT analysis was performed on 96-well culture plates, and colorimetric analysis was conducted to reduce 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) to blue formazan product. Based on the DMSO was added to each 96-well plate after 2 hours of MTT (1 mg / ml) incubation at 37 ° C. The absorbance of the soluble MTT formazan product was measured spectrophotometrically at 570 nm.

In the results of cell adsorption rate, at the weak CNT / CNF concentration, each nanomaterial had little effect on cell adsorption. However, after 3 hours after the injection of 5ug / ml shows the same result as Figure 1, it can be seen that as the concentration increases the cell adsorption is also affected. But from a statistical point of view (AVOVA analysis), there was not much difference.

< Experimental Example  2> Carbon nanomaterials  Neurotumor Cells On growth  Impact experiment

Each nanomaterial (mwCNT, phCNF, hbCNF, hlCNF) was treated to cells by concentration and time, and MTT analysis was performed. mwCNT had the strongest cytotoxicity by concentration, and phCNF was also cytotoxic but weaker than mwCNT. hbCNF is rarely toxic (FIG. 2A).

MTT assay was performed for each hour after treatment with carbon nanomaterial (5 ㎍ / ml). Cells increase MTT value over time through division, and when the carbon nanomaterials are treated, the value decreases. In particular, after 24 hours, mwCNT and hlCNT had values of 40.286% and 37.182%, respectively, compared to after 24 hours of control, and after 81 hours, only 81.992% and 118.529% of the values were shown. This shows that mwCNT and hlCNF are more cytotoxic than other carbon nanomaterials of the same size (FIG. 2B).

Experimental Example 3 Correlation Examination of Carbon Nanomaterials and Apoptosis

200000 U373MG cell lines were each dispensed into 6-well plates, incubated for 48 hours with a culture medium containing 5 μg / ml of nanomaterials, and TUNEL analysis was performed.

TUNEL assay was centrifuged cells washed with PBS / BSA and fixed for 15 minutes with paraformaldehyde (paraformaldehyde). And permeate at Triton-X 100. Thereafter, FITC-conjugated dUTP was treated using an apoptosis detection system kit (Roche, Mannheim, Germany. Cat. 11 684 817 910) and observed by fluorescence microscopy.

TUNEL-positive cells, as seen in the TUNEL assay, detect free 3'-OH groups when DNA strands fragment, suggesting cell death.

As a result, no apoptosis was observed in any experimental group (FIG. 3).

Western blots were also performed to observe at the protein level.

Cell lines were lysed with lysis buffer (25 mM HEPES; pH 7.4, 100 mM NaCl, 1 mM EDTA, 5 mM MgCl ₂ , 0.1 mM DTT and protease inhibitor mixture). Proteins were placed on a 12.5% SDS (sodium dodecyl sulfate (SDS)) gel with 15 μg of protein per well and electrophoresed at 100 volts for 1 hour 40 minutes. After the electrophoresis of the SDS gel was in close contact with the nitrocellulose membrane, the proteins immobilized on the SDS gel were transferred to the nitrocellulose membrane for 30 minutes at a current of 15 mA. After the protein transfer membrane was washed with TBS (tris buffered saline), it was pretreated with 5% nonfat dry milk and reacted with caspase-3 (cell signaling. Cat. 9661) antibody at a concentration of 1: 1000 for 16 hours. Rinsed with TBS containing 0.1% Tween, and then reacted with a rabbit secondary antibody at a concentration of 1: 10000 to which horseradish peroxidase was conjugated for 1 hour. Finally, the fluorescent reaction was performed with ECL reagent, and the immune response was detected by an image analysis system (LAS-4000, Fujifilm).

Caspase-3 is a cell apoptosis marker protein. When apoptosis occurs, the protein is cut to 19 kD.

No cut caspase-3 was detected in all experimental groups (FIG. 3).

Experimental Example 4 Influence of Carbon Nanomaterials on Cell Migration and Proliferation

5 ㎍ / ml of carbon nanomaterials were treated in U373MG cells and observed live using a Cell Observer (Carl Zeiss, Germany) to investigate cell migration and division. Cell Observer satisfies all the conditions for culturing cells (CO ₂ , temperature, humidity), and takes a picture of the cell in seconds or minutes to make it into a video and analyze the results. In this experiment, images were taken and analyzed for 7 or 24 hours in increments of 5 minutes. As shown in Figure 4a it was possible to measure the path of all the cells moving from the position of the first cell to the last position. Through this, the movement speed and movement distance of the cells (Fig. 4b) can be measured, and the number of divisions in the video (Fig. 4c) was also measured.

As a result, as shown in Figure 4b it was confirmed that all carbon nanomaterials affect the movement distance and speed of the cells.

In addition, FIG. 4C, which analyzed cell division, indicates that the nanomaterial is also involved in the proliferation of cells, and the results were similar to those of the MTT results.

Experimental Example 5 Effect of Carbon Nanomaterials on HT22 and Neuronal Cells

MTT analysis was performed by treating the hematopoietic stem cell line (HT22) with 5 μg / ml of carbon nanomaterial before differentiation into neurons.

As a result, as a result of treating each carbon nanomaterial to HT22 cells before they were differentiated into neurons, it was confirmed that they showed the same pattern as neuronal tumor cells (FIG. 5).

However, when HT22 cells were differentiated, that is, when each carbon nanomaterial was treated to neurons that did not divide any more and completed differentiation, there was no toxicity. This strongly supports the foregoing results that carbon nanomaterials do not participate in cell death and affect division (FIG. 6).

Claims (8)

Cancer cell proliferation inhibiting composition comprising the carbon nanomaterial as an active ingredient.
The composition of claim 1, wherein the carbon nanomaterial is multi-walled carbon nanotubes or nanofibers.
The composition of claim 2, wherein the multi-walled carbon nanotubes have a diameter of 10 nm to 50 nm.
The composition of claim 1, wherein the carbon nanomaterial is dispersed in serum medium and treated in cancer cells.
The composition of claim 1, wherein the composition is a pharmaceutical composition.
The composition of claim 1, wherein the carbon nanomaterial is included in an amount of 0.01 to 10 μg / ml.
The composition of claim 1, wherein the cancer cells are brain tumor cells that continue to proliferate.
The composition of claim 1, wherein the composition is injected in the form of an injection into a cancer cell development site.

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