KR20140013390A - Evaluation method of toxicity of nanoparticles - Google Patents

Abstract

The present invention relates to a method for evaluating toxicity of nanoparticles, and more particularly, to a method for evaluating toxicity of nanoparticles comprising the steps of: obtaining a sample of tissues or cells of a mammal exposed to nanoparticles; irradiating at least one selected from a group consisting of a glutamic acid content, a pyruvate content, whether mitochondria within a cell is damages or not, a reactive oxygen species (ROS) content and an ATP content within the sample; comparing with a control group. The method for evaluating toxicity of nanoparticles according to the present invention analyzes factors having high a correlation with the toxicity of nanoparticles. By using this, it is possible to more precisely detect the toxicity of nanoparticles with excellent detection sensitivity, and through this, it can be effectively used in monitoring or evaluating harmfulness of the toxicity of nanoparticles, as well as effectively used as a tool for examining various diseases, the effects on health or the like when exposed to nanoparticles.

Description

Evaluation Method of Toxicity of Nanoparticles

The present invention relates to a method for evaluating toxicity of nanoparticles.

The development of nanotechnology is becoming more and more available not only for the industrial use of nanomaterials but also for medical and research purposes. As a result, not only workers producing nanomaterials but also the general public are increasingly exposed to and exposure to nanomaterials.

However, although nanotechnology offers many benefits and benefits that are perceived as a new technological revolution throughout the industry, it is also a well-known fact that these potential risks It can be attributed to the characteristics of nanotechnology.

That is, the smaller the particle, the larger the ratio of the specific surface area. Small particles having such a large specific surface area ratio increase the toxicity when reacted with the biotissues. For example, some nanoparticles such as titanium dioxide, carbon powder, It has already been revealed through academic experiments that the smaller the size, the more toxic it becomes, such as inflammation. In addition, ultrafine nanoparticles may not penetrate into the airways or mucous membranes but may penetrate deep into the alveoli or migrate to the brain. Moreover, recent studies have shown that nanoparticles accumulate in the body to cause diseases or central nervous disorders .

Therefore, in recent years, along with the development of nanotechnology, stability evaluation of nanotechnology has also been actively carried out, mainly focusing on researches of devices for evaluating toxicity of nanoparticles (Korean Patent Registration No. 10-0798149) There are no clear methodological guidelines or biologic indices that can be used to assess the safety of various nanomaterials.

Therefore, the present invention aims to provide a biological indicator for the functional change of metabolites and cells as an approach to evaluate the safety of nanomaterials using Sys Biological Analysis Method.

In order to solve the above problems, the present invention comprises the steps of obtaining a tissue or cell sample of a mammal exposed to nanoparticles; Irradiating any one or more selected from the group consisting of glutamic acid content, pyruvate content, intracellular mitochondrial damage, ROS (active oxygen species) content, and ATP content in the sample; And comparing with a control; provides a method for assessing the toxicity of nanoparticles comprising a.

Toxicology evaluation method of the nanoparticles according to the present invention by analyzing the factors having a high correlation with the toxicity of the nanoparticles, by using it can more accurately determine whether the toxicity of the nanoparticles with excellent detection sensitivity, through which the harmfulness of the nanoparticles It can be useful for monitoring or evaluating the effects of the disease, and can be useful as a tool for identifying various diseases or health effects caused by exposure to nanoparticles.

FIG. 1 shows the result of a cytotoxicity study of MNPs @ SiO ₂ (RITC) according to Example 1. FIG.
FIG. 2 shows the results of analysis of gene expression changes of HEK 293 cells treated with MNPs @ SiO ₂ (RITC).
Figure 3 shows the amino acid change analysis of HEK 293 cells treated with MNPs @ SiO ₂ (RITC).
FIG. 4 shows the result of selective ion detection (SIM) chromatogram analysis of HEK 293 cells treated with MNPs @ SiO ₂ (RITC).
Figure 5 is a schematic diagram of the metabolic pathway of glutamate built through elaborate career analysis (IPA).
FIG. 6 shows PCR results showing changes in gene expression of MNPs @ SiO ₂ (RITC) -treated HEK 293 cells.
FIG. 7 shows the results of ROS content analysis of HEK 293 cells treated with MNPs @ SiO ₂ (RITC).
Figure 8 is an electron micrograph for mitochondrial damage analysis of HEK 293 of the MNPs @ SiO ₂ (RITC) treated cells.
FIG. 9 shows the results of comparing the ATP content of HEK 293 cells treated with MNPs @ SiO ₂ (RITC).
FIG. 10 shows the results of analyzing changes in organic acids (OA) in the Krebs cycle using H-293 cells treated with MNPs @ SiO ₂ (RITC) using GC-MS.
FIG. 11 is a schematic diagram showing a change in intracellular total mechanism due to the nanoparticle treatment according to the present invention. FIG.

The present invention comprises the steps of obtaining a tissue or cell sample of a mammal exposed to the nanoparticles; Irradiating any one or more selected from the group consisting of glutamic acid content, pyruvate content, intracellular mitochondrial damage, ROS (active oxygen species) content, and ATP content in the sample; And comparing with a control; provides a method for assessing the toxicity of nanoparticles comprising a.

The inventors of the present invention while studying to evaluate the harmful effects of exposure to nanoparticles through changes in cells or tissues caused by nanoparticles, the concentration of glutamic acid in the cells when a certain concentration of nanoparticles are treated, cell samples Increased ROS content, decreased ATP content, damaged intracellular mitochondrial lining, increased glutamic acid content and pyruvate content. It confirmed and completed this invention.

In one embodiment of the present invention, the nanoparticles may be all nanoparticles having increased toxicity when reacted with biotissues. More specifically, the nanoparticles may be atmospheric nanoparticles, nanoparticles contained in a cosmetic composition, Including, but not limited to, nanoparticles, semiconductor nanoparticles, and the like. The nanoparticles may also be in the form of a core-shell, and the shell may include, but is not limited to, silica.

In one embodiment of the present invention, the method for evaluating the toxicity of the nanoparticles may include essentially examining the glutamic acid content or pyruvate content.

When the amount of glutamic acid or pyruvate is increased compared to the control group, the exposed nanoparticles may be evaluated to possess toxicity. Glutamic acid content and pyruvate content may be measured by GS-MS analysis, but are not limited thereto.

In addition, the ROS content can be measured by fluorescence analysis using 2 ', 7'-dichlorodihydrofluorescin diacetate (DCFH-DA) dye. For the ATP content, the luminescence characteristics are analyzed using ATP analysis system (Promega, USA) But is not limited thereto. If the ROS content is higher than that of the control, the nanoparticles can be evaluated as possessing toxicity. If the ATP content is lower than that of the control, the nanoparticles can be evaluated as having toxicity.

In addition, the damage of the mitochondria can be analyzed by electron microscopy. When the mitochondrial lining tissue is damaged as shown in the embodiment of the present invention and 8 can be evaluated that the nanoparticles retain toxicity.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Experimental Example >

The following experimental examples are intended to provide experimental examples that are commonly applied to each embodiment according to the present invention.

1. Cell culture and MNPs @ SiO ₂ ( RITC ) process

The human embryonic kidney cell 293 (HEK 293) cell line has been well known for its relevance to the cytotoxicity induced by silica nanoparticles and its relation to the human renal toxicity model, and MNPs @ SiO ₂ (RITC) The toxicity of the particles was evaluated. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco, USA), 100 units / ml penicillin, 100 ug / mL streptomycin (Gibco, USA) Lt; / RTI &gt; The cells were cultured at 37 ° C in 5% humidity, CO ₂ And cultured under atmospheric conditions. The medium was replaced every 3 days. The MNPs @ SiO ₂ (RITC) particles having a diameter of 50 nm was added directly to the culture medium without serum and antibiotics. HEK 293 cells were seeded at a concentration of 2 x 10 ⁶ cells / 100 mm and cultured for 12 hours in 10 ml of medium containing various concentrations of MNPs @ SiO ₂ (RITC). Cells were washed twice with PBS (phosphate buffered saline), and harvested using 0.25% trypsin / 0.1% EDTA (Sigma, USA).

2. FACS ( Fluorescence activated cell sorting ) analysis

FACS analysis was performed using Annexin V-FITC Apoptosis Detection Kit (Becton Dickinson Biosciences, USA) for apoptosis analysis.

More specifically, the cells collected after the incubation were washed twice with cold PBS and resuspended in 100 μL of 1 × binding buffer (10 mM Hepes, pH 7.4, 140 mM sodium chloride, and 2.5 mM calcium calcium chloride). 5 μL of Annexin V-FITC and propidium iodide (PI) were then added to the sample and incubated for 15 minutes at 25 ° C under dark conditions. To stop the reaction, 400 [mu] L of 1X binding buffer was added to each tube. Cells were analyzed by FACS (Becton Dickinson Facsvantage, USA) and data analysis was performed using Win MDI Version 2.9 (The Scripps Research Institute, USA).

3. Confocal laser scanning microscopy  ( LSM ) And imaging

HEK 293 cells treated with MNPs @ SiO ₂ (RITC) were fixed in 4% formaldehyde solution and fixed with anti-fade solution (R & D, USA) after dehydration. Cells were also stained with 4 ', 6-diamidino-2-phenylindole (DAPI) for nuclear detection. Fluorescence images were acquired using confocal LSM (Olympus, Japan). DAPI and MNPs @ SiO ₂ (RITC) were excited at wavelengths of 205 and 530 nm, respectively. Digital images were analyzed using LSM software (Leica, USA).

4. RNAzol  Using B RNA  refine

RNA of HEK 293 cells treated or not treated with MNPs @ SiO ₂ (RITC) was isolated using RNAzol B (Tel-Test, Inc., USA) and purified using RNeasy Mini Kit (Qiagen, USA) . More specifically, the harvested cells were treated with 1 ml of RNAzol B, followed by addition of chloroform and incubation at 4 ° C for 5 minutes. Total RNA was precipitated using 600 [mu] l of iso-propanol and the RNA pellet was washed with 70% ethanol. RNA was eluted from the pellet using RNase-free water (WelGene, Korea) and quantitated by spectrophotometer (Eppendorf, USA) and agarose gel electrophoresis. The purity of the RNA used in the microarray and PCR experiments was 1.9-2.0 based on optical density (OD) 260/230 and 260/280.

5. qRT - PCR  And real time PCR

To quantify gene expression, total RNA samples were reverse transcribed using the ImProm-II Reverse Transcription System (Promega, USA) and amplified using primers for each fragment. The amplified PCR products were separated on 1% agarose gel and stained with ethidium bromide. The amount of amplified PCR product was calculated using Multi Gauge 3.0 software (Fujifilm, Japan). The expression levels of glutamic acid-related genes were analyzed with a gene-specific primer set using RealMOD ™ SYBR Green Real-time PCR kit (iNtRON Biotechnology, Seongnam, Korea) and DNA Engine (Bio-Rad, Hercules, Calif.). The reaction was carried out at 5 ° C for 2 minutes, at 95 ° C for 30 seconds, then at 95 ° C for 5 seconds and at 57 ° C for 30 seconds. PCR products were analyzed by forming melting curves using a MJ Opticon Monitor Version 3.1 (Bio-Rad, Hercules, Calif.).

6. Microarray  Experiment and data analysis

To construct the gene expression profile, total RNA was hybridized to a microarray gene chip (Affymetrix, USA). More specifically, the Affymetrix system (Beyond Bioinformatics ISTECH AATC) Human U133 Plus 2.0 50 K microarray containing 54,675 gene specific probes was analyzed and hybridization was carried out overnight using 15 μg of the labeled RNA product. After hybridization, the array was washed several times and scanned with an Affymetrix scanner. Pretreatment was performed using GCOS, Global scaling in GenPlex 3.0 software (ISTECH, Korea). Gene expression changes were analyzed by MA-plot of control cells and experimental arrays. The genes whose expression level changed by more than 1.25 times were examined by Ingenuity Pathway Analysis (IPA Ver. 8.5, Ingenuity Systems, http://www.ingenuity.com ).

7. Intracellular AA  Sample preparation for profile analysis

AA of the control HEK 293 cells treated with no nanoparticles and the experimental cells treated with 0.1 and 1.0 μg / μL were detected by GC-MS. Specifically, the pH of each sample (1 x 10 ⁵ cells) after the addition of IS (International Standard, Norvalin, 200 ng) was adjusted to pH ≥ 12 and the ECF of dichloromethane phase (1 mL) (ethyl chloroformate, 20 μL ) and vortexing were performed. The reaction mixture was extracted with diethyl ether (3.0 mL) and ethyl acetate (2.0 mL), which was evaporated under nitrogen reflux (40 ° C). The residue was reacted with MTBSTFA (20 [mu] L) in toluene (20 [mu] L) for 30 min at 60 [deg.] C for GC-SIM-MS.

8. Gas Chromatography-Mass Spectrometry

GC-MS analysis was performed using an Agilent 5973 mass-selective detector (70 eV, electron impact mode) as an interface with the Agilent 6890 gas chromatograph, Ultra-2 (5% phenyl-95% methylpolysiloxane bonded phase; 25 mx 0.20 mm id , 0.11 μm film thickness) crosslinked capillary column (Agilent Technologies, USA). The injector, interface, and ion source were 260, 300, and 230 ° C, respectively. A constant flow rate of 0.5 mL / min of helium was used as the mobile gas, and samples were injected in a split-injection mode (10: 1). The temperature for AA (amino acid) analysis was set at 140 ° C for the initial 2 minutes, and the temperature was raised at a rate of 5 ° C / min. At 240 ° C. Then, the temperature was raised to 300 ° C at a rate of 30 ° C / Respectively. The temperature for the OA (organic acid, organic acid) analysis was set at 100 ° C for the initial 2 minutes. After the temperature was raised to 250 ° C at 5 ° C / minute, the temperature was raised to 300 ° C at 20 ° C / Respectively. The mass range was 50-600u and the scan speed was set at 0.99 scans / s. In the SIM mode, three characteristic ions of each AA were used for peak detection and quantification.

9. Transmission electron microscopy observation

Cells obtained via trypsin / EDTA and washed twice with PBS were fixed in Karnovsky's fixative solution (1% paraformaldehyde, 2% glutaraldehyde, and 2 mM calcium chloride) for 48 hours and then washed with chocodilate buffer. The cells were then incubated in 1% osmium tetroxide (OsO ₄ ) solution containing 0.05% potassium ferrocyanide (K ₄ Fe (CN) ₆ ) for 90 minutes and fractionated using Reichert Jung Ultracul S (Leica, USA). The cells were stained with uranyl acetate and lead citrate and then observed at arbitrary sites of the sample at x 25,000 magnification using a TEM (JEM-1400, JEOL LTD., Japan).

10. ROS ( Active oxygen species ) Measure

Intracellular ROS was measured via 2 ', 7'-dichlorodihydrofluorescin diacetate (DCFH-DA) dye. Specifically, 1 x 10 ⁴ and 2 x 10 ⁴ cells were dispensed into chamber slides and black 96-well plates, respectively, and MNPs @ SiO ₂ (RITC) (0.1 and 1.0 μg / μL) were treated for 12 hours. After the medium was removed, the cells were washed twice with PBS buffer. 1x DCFH-DA / media solution was then added to each well and incubated at 37 ° C for 1 hour. The sample was washed twice with PBS and fluorescence was measured with confocal LSM (Olympus, Japan) and Gemini EM Fluorescence Microplate Reader (Molecular Devices, USA) under 480 nm excitation / 530 nm emission conditions. When cell nuclei were detected using confocal LSM, cells were stained with DAPI.

11. ATP  Concentration measurement

ATP concentrations in cells were measured using an ATP assay system (Promega, USA). Specifically, the cells were obtained with 0.25% trypsin / 0.1% EDTA, washed twice with PBS, resuspended in 0.1% trichloroacetic acid (TCA, Sigma, USA) and incubated at room temperature for 10-30 minutes. The luciferin reagent was mixed with the reconstitution buffer solution, and the resuspended sample was treated with 50 μL of luciferin reagent / buffer mixture and dispensed into a 96 well cell culture plate. After incubation for 10 minutes at 25 캜, the luminescence properties of each well were measured with LMax? ³⁸⁴ (MDS Analytical Technologies, USA) and imaged using LAS-1000 (FUGIFILM, Japan).

< Example  1 > MNPs @ SiO ₂ ( RITC ) Cytotoxicity studies

In order to examine the cytotoxicity, 0.01, 0.1, and 1.0 μg / μL of MNPs @ SiO ₂ (RITC) were treated for 12 hours in HEK 293 cells and morphological changes were observed. The concentration range of the MNPs @ treated cells SiO ₂ (RITC) was the morphological changes observed in the cell membrane, or organelle structure (see FIG. 1A). To confirm the viability of cells according to the two concentration ranges (0.1 and 1.0 μg / μL), the samples were labeled with Annexin V and PI and subjected to FACS analysis. The results are shown in FIG. 1B and FIG. Cell viability was 99.34 and 98.69% at concentrations of MNPs @ SiO ₂ (RITC) 0.1 and 1.0 μg / μL, respectively, and there was no significant difference compared to the control group. In addition, the MTS assay was performed to evaluate cytotoxicity after 3 or 7 days of treatment with MNPs @ SiO ₂ (RITC) 0.1 or 1.0 μg / μL, and there was no significant difference compared to the control. The results show that treatment of MNPs @ SiO ₂ (RITC) mothamyeo not affect the survival rate of the HEK 293 cells, thus MNPs @ SiO of cell growth and death is the range of concentrations (0.01, 0.1, and 1.0μg / μL) ₂ ( RITC) was not affected.

< Example  2 > MNPs @ SiO ₂ ( RITC ) Has been processed HEK  Gene expression of &lt; RTI ID = 0.0 &gt; AA  Composition analysis

MNPs @ SiO to confirm that ₂ (RITC) is increasing or decreasing the expression of any gene in the treated HEK 293 cells, and 0, 0.1, and the gene of 1.0 μg / μL in MNPs @ SiO ₂ (RITC) is the HEK 293 cells were treated Expression assays were performed using an oligonucleotide microarray. After standardizing gene expression, 5,966 functional gene probes were categorized into eight clusters using GenPlex 2.0 software. Clusters 1 and 3 showed a large signal change, which was analyzed more specifically. When 1.25 fold was set as the cutoff value, 291 genes out of the 466 genes in the cluster showed an increase or decrease in expression due to 1.0 μg / μL MNPs @ SiO ₂ (RITC), which is shown in FIG. 2A. The signal intensities of the total functional genes contained in clusters 1 and 3 were plotted according to the intensity level difference of the group, and the plot-pattern was separated into high expression and low expression groups (see FIG. 2B). Differences in the expression levels of the nanoparticle treated group and the control group were found to be related to the treatment concentration of MNPs @ SiO ₂ (RITC). Cells treated with 0.1 μg / μL of MNPs @ SiO ₂ (RITC) showed a 7.8% increase in gene expression and a 1.4% decrease in gene expression compared to the control, while 1.0 μg / μL MNPs @ SiO ₂ (RITC) Treated cells showed a 22.0% increase in gene expression and a 35.8% decrease in gene expression compared to the control. The inventors of the present invention classified the genes that did not change in the treatment group of 0.1 μg / μL nanoparticles, although the expression level was remarkably changed in the 1.0 μg / μL nanoparticle-treated group, which is shown in Table 1. Table 1 classifies the 291 gene of the above-mentioned condition as a function, and it includes a gene having increased expression of 205 and a gene having decreased expression of 86.

[Table 1]

Referring to Table 1, the inventors of the present invention performed AA profile analysis, as the metabolism-related gene expression was shown to decrease due to the treatment of MNPs @ SiO ₂ (RITC). The AA composition of the cells treated with the control, 0.1 μg / μL, and 1.0 μg / μL MNPs @ SiO ₂ (RITC) is shown in Table 2 and the AA composition change compared to the mean value of the control group is shown in FIG. .

[Table 2]

Referring to Table 2 and FIG. 3, it was confirmed that the treatment of MNPs @ SiO ₂ (RITC) with 1.0 μg / μl significantly increased the expression of glutamic acid (about 200%), while the other amino acids , Leucine, isoleucine, proline and tyrosine) decreased (? 30%). On the other hand, the group treated with 0.1 μg / μL MNPs @ SiO- ₂ (RITC) showed little change (increase / decrease of 20% compared to the control).

Selective ion detection (SIM) chromatograms also showed a large increase in glutamic acid expression in 1.0 μg / μL MNPs @ SiO ₂ (RITC) treated cells (see FIG. 4). 4, the relative proportion of the peak area of glutamic acid to internal standard (IS) was 0.056 (FIG. 4A; control), 0.045 (FIG. 4B; 0.1 μg / μL MNPs @ SiO ₂ (RITC) 4C; 1.0 μg / μL MNPs @ SiO ₂ (RITC)). In particular, the levels of glutamic acid in cells treated with 1.0 μg / μL MNPs @ SiO ₂ (RITC) were significantly higher than in the control and 0.1 μg / μL treated groups.

The branched-chain amino acids (valine, leucine and isoleucine) play an essential role in the synthesis of the immune system and glutamate, and over 30% of glutamic acid is synthesized from BCAA through the BCAA transaminase pathway. Table 2 and according to FIG. 3, bar, the observed changes in the BCAA suggesting dropped below this the amino acid changes affect the expression of the glutamic acid due to MNPs @ SiO ₂ (RITC).

< Example  3 > MNPs @ SiO ₂ ( RITC ) Processing Sophisticated Care Analysis of Glutamic Acid Metabolism Ingenuity pathway analysis , IPA )

The results of GC-MS showed that the level of glutamic acid was increased more than two times by treatment with 1.0 μg / μL MNPs @ SiO ₂ (RITC). Thus, the inventors of the present invention A genetic network of metabolic pathways of glutamate was constructed through sophisticated career analysis (IPA) software.

The metabolic pathway of glutamic acid was constructed based on the gene whose expression level changed (fold change > 1.25) due to the treatment of 1.0 μg / μL MNPs @ SiO ₂ (RITC) and its metabolism, which is shown in FIG.

(GAD1), glutamic acid dehydrogenase 1 (GAD1), glutamic acid dehydrogenase 1 (GAD1), and glutamic acid dehydrogenase 1 (GAD1) The gene is included in the glutamate metabolism pathway, indicating that it is an increased or decreased gene in cells treated with 1.0 μg / μL MNPs @ SiO ₂ (RITC). Therefore, the above results suggest that glutamic acid expression changes are caused by various gene expression changes associated with the glutamic acid metabolism pathway.

< Example  4> semi - quantitative PCR Analysis of Gene Expression Associated with Glutamic Acid Metabolism Pathway

Associated genes involved in catalyzing the conversion of glutamic acid to other metabolic precursors or amino acids, such as GLUD1, GOT2, GAD1, and GLUL, were reduced in expression by treatment with 1.0 μg / μL MNPs @ SiO ₂ (RITC) 6A). In particular, expression of GLUD1 was reduced by 47% compared to the control, and GOT2, GLUL, and GAD1 decreased by 33, 25, and 61%, respectively. On the other hand, in the cell group treated with 0.1 μg / μL MNPs @ SiO ₂ (RITC), there was no significant change in the expression level compared to the control group.

On the other hand, the expression of GPT2 and ALDH2H4A1, which are known as genes that induce the production of glutamic acid by catalyzing the conversion of amino groups of alanine and proline, is increased by about 41 and 53% in 1.0 μg / μL MNPs @ SiO ₂ (RITC) (See FIG. 6B). These results are in agreement with the results (see FIG. 3) in which GC-MS analysis showed that levels of alanine and proline decreased while levels of glutamic acid increased with the treatment of 1.0 μg / μL MNPs @ SiO ₂ (RITC) Suggesting that overexpressed GPT2 and ALDH4A1 genes are involved in the expression results.

< Example  5> active Oxygen species  Generation and Mitochondrial Damage Analysis

Since many oxidative stresses due to nanoparticles have been reported in the existing literature, it was analyzed whether oxidative stress caused by MNPs and damage to mitochondria caused by them.

The increase of intracellular reactive oxygen species (ROS) was analyzed by treatment of MNPs @ SiO ₂ (RITC) with 2 ', 7'-dichlorodehydrofluorosyntioacetate (DCFH-DA) Figure 7 shows the result of visualization with confocal LSM.

Referring to FIG. 7A, it was confirmed that the concentration of active oxygen increases in the treatment group of 1.0 μg / μL MNPs @ SiO ₂ (RITC) (FIG. 7A). It can be confirmed that the amount of oxygen is increased by about 63% when compared with the control group (FIG. 7B). On the other hand, in the treatment group of 0.1 μg / μL MNPs @ SiO ₂ (RITC), there was no change in ROS as compared with the control group.

< Example  6> ATP  Synthetic and Krebs cycle Of organic acids associated with

The damage of mitochondria caused by the active oxygen was analyzed by an electron microscope (TEM) and a JC-1 assay kit (Molecular Probe, USA). The result is shown in FIG. Referring to FIG. 8, it was confirmed that the endothelial tissue of mitochondria was damaged in the treatment group of 1.0 μg / μL MNPs @ SiO ₂ (RITC)

On the other hand, no significant change was observed with the control cells in the treatment group of 0.1 μg / μL MNPs @ SiO ₂ (RITC).

The main function of the mitochondria is the production of adenosine triphosphate (ATP), the main energy source of the cell through the Krebs cycle, which is predicted to be altered by damaged mitochondria. Therefore, the amount of ATP synthesis was measured by a fluorescence-based ATP concentration analysis system.

As a result, there was no significant difference between the control group and 0.1 μg / μL MNPs @ SiO ₂ (RITC) treatment group, but the production of ATP was decreased in the treatment group of 1.0 μg / μL MNPs @ SiO ₂ (RITC) (See Fig. 9A). Further, as a result of quantifying the fluorescence image, ATP reduction amount was about 50% as compared with the control group (see FIG. 9B). These results suggest that ATP production by the damaged mitochondria was also reduced by treatment with 1.0 μg / μL MNPs @ SiO ₂ (RITC).

The ATP of the cell is produced mainly through the Krebs cycle in the mitochondria, and the Krebs cycle is known as the complex chain metabolism of organic acids such as pyruvate, α-ketoglutarate and oxaloacetate. The reduction of ATP implies the abnormal activity of the Krebs cycle, and the change of organic acids (OA) in the Krebs cycle was analyzed using GC-MS. The results are shown in Fig. Referring to FIG. 10, in the 1.0 μg / μL MNPs @ SiO ₂ (RITC) -treated group, pyruvate was increased by 50% or more, while the expression of major organic acids in the Krebs cycle, such as α-ketoglutarate and oxaloacetate, 17% and 14%, respectively. It was also found that the fumarate and maleate, which are other organic acids in the other Krebs cycle, also decreased due to 1.0 μg / μL MNPs @ SiO ₂ (RITC) treatment.

Treatment with MNPs at a high concentration (1.0 μg / μL) could induce a change in gene expression, especially the expression of metabolic machinery. This led to a change in metabolism and a significant change in glutamate. In addition, active oxygen induced by high concentrations of MNPs showed mitochondrial damage and ATP reduction, and it was also found that Krebs cycle related organic acids could be changed. On the other hand, treatment with low concentrations of MNPs showed similar results to the control group, showing no significant change in gene and metabolism. The mechanism is summarized in FIG.

Therefore, high concentration of nanoparticles can affect the living body in terms of genetic and metabolism. A clear methodological guideline for evaluating the safety of nanoparticles at present and a method for clearly evaluating the nanoparticles through the present invention in the situation where biological indicators are not standardized are presented.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that such detail is solved by the person skilled in the art without departing from the scope of the invention. will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (5)

Obtaining a mammalian tissue or cell sample exposed to the nanoparticles;
Within the sample
Glutamic acid content,
Pyruvate content,
Intracellular mitochondrial damage,
ROS (active oxygen species) content, and
Irradiating any one or more selected from the group consisting of ATP content; And
Comparing with a control; To evaluate the toxicity of nanoparticles comprising a. The method of claim 1,
Toxicity evaluation method of the nanoparticles
How to evaluate the toxicity of the nanoparticles which comprises essentially the step of examining the glutamic acid content or pyruvate content. The method of claim 1,
The nanoparticles are nanoparticles in the atmosphere, nanoparticles contained in the cosmetic composition, nanoparticles contained in the pharmaceutical composition or semiconductor nanoparticles is a method for assessing the toxicity of nanoparticles. The method of claim 1,
Wherein the ATP content is reduced by the exposure of the nanoparticles toxicity evaluation method of nanoparticles. The method of claim 1,
Wherein the ROS content, glutamic acid content and pyruvate content is increased by the exposure of the nanoparticles to evaluate the toxicity of the nanoparticles.

Images