KR20120102180A - An in-vitro method for the toxicity assessments of nano-materials - Google Patents

Abstract

PURPOSE: A method for analyzing nano material risk is provided to minimize the effect by non-homogeneity of nanoparticles of a solution and to accurately evaluate the nano material risk. CONSTITUTION: A method for evaluating nano material risk in vitro comprises: a step of attaching a cell layer on the surface of a substrate; a step of exposing nano materials to the cell layer and dyeing using a normal and inverted exposure device; a step of analyzing the cell layer; and a step of quantitating cell apoptosis degree. The cell layer is formed on a microfluidic cell chip, a multiwell, or slide glass. The normal and inverted exposure device includes the microfluidic chip or wellplate exposing device made of polymer materials of [poly(dimethylsiloxane)](PDMS), polymethylmethacrylate(PMMA), polyacrylates, polycarbonates, polycyclic olefins, polyimides, or polyurethanes.

Description

An in? Vitro Method for the Toxicity Assessments of Nano? Materials}

The present invention relates to a nanomaterial risk assessment method, and more particularly, using a reverse exposure mechanism, and optionally by using a cell-specific image cytometry (Flow cytometry) or flow cytometry, improved error, in in vitro An accurate method for assessing nanomaterial risk.

The risk of nanomaterials that can occur when the human body is exposed to nanoscale particles (hereinafter referred to as nanomaterials) is emerging as a new problem due to the recent rapid development of nanotechnology. Given the increasing frequency of inhalation by breath, inhalation by mouth, and skin exposure, accurate and scientific information on the stability of nanotechnology and nanomaterials is urgently required.

The risk of nanomaterials is due to their very small size and the physicochemical properties of particles that differ significantly from the bulk material specific to nanomaterials. Hazards such as the likelihood of causing cancer of needle-like materials such as asbestos and fiberglass have been known for a long time. Unlike asbestos and glass fibers, nanomaterials are attracting more attention because of their small size as well as their shape characteristics. It is reported that high surface reaction force and cell membrane permeability allow nanomaterials to easily enter the living body, increase the possibility of causing stress at the cellular level, and furthermore, accumulate in the living body like asbestos and have a lasting effect. Nanomaterials can affect cardiovascular disease because they can penetrate deeper into the body and deposit them, unlike micro-sized materials. Especially, nanomaterials penetrated through the nasal nerves can be moved in the body by blood. It may also affect brain damage.

Most of the risk studies of nanomaterials in vivo are carried out through injection or cell culture, mainly targeting metals, metal oxides, and carbon nanomaterials. In general, nanoparticles are known to enter the human body by respiratory, oral, and skin rather than injection.

International cooperation activities should be actively promoted to prevent and prepare for potential harmful effects of nanomaterials, while at the same time institutional arrangements to minimize or reduce potential risks such as indiscriminate nanotechnology development and application and inappropriate disposal. It's time to get it done.

However, contrary to the fact that nanoparticles and the like are considered to be seriously harmful to humans or the environment, even actual exposure of humans and ecosystems by nanomaterials, toxicity and harmful effects assessment methods are not properly established.

To date, risk assessment methods commonly used in conventional chemicals are often applied to nanomaterials. However, the smaller the size of the particles, the larger the surface area of the responsiveness and subsequent toxicity increases in biological tissues, it is important to investigate the new physicochemical properties of the nanomaterials and their toxicity considering the characteristics of the nano-size.

Therefore, the risk assessment of nanomaterials requires the study of the unique properties and problems of nanoparticles, their potential effects on behavior, exposure and toxicity.

Accordingly, the present inventors have used the inverted exposure mechanism to compensate for the problems of the previously reported nanotoxicity evaluation in assessing the cytotoxicity of the nanoparticles. Minimized, and confirmed that the error of the conventional cell death assay caused by the optical or catalytic reactivity of the nanoparticles by using cell-specific image analysis (Image Cytometry), and completed the present invention.

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for evaluating the risk of nanomaterials in vitro using a method of upright and inverted exposure and image analysis by cell.

It is another object of the present invention to provide a method for evaluating the risk of nanomaterials in vitro using a method of upright and inverted exposure and flow cytometry.

It is another object of the present invention to provide a flow cytometry platform or system for risk assessment of nanomaterials for using the method.

In order to solve the above problems, the present invention

Attaching a cell layer to the surface of the substrate;

Exposing and staining the nanomaterials in the cell layer using upright and inverted exposure apparatuses;

Performing an analysis on the cell layer; And

It provides an in vitro nanomaterial risk assessment method comprising the step of quantifying the degree of apoptosis by the analysis result. .

In this case, the cell layer may be formed in a microfluidic cell chip, a multi well or a slide glass, and may be used by forming a single or multiple cell layers.

The use of the upright and inverted exposure apparatus includes concentration correction due to heterogeneity in the cell medium of the nanomaterial. That is, while exposing the nanomaterial to the cell layer by using the upright and inverted exposure apparatus, concentration correction due to inhomogeneity in the cell medium of the nanomaterial is performed. At this time, the concentration of the exposed nanomaterials can be corrected by modifying and using known formulas (theoretical and experimental formulas) reflecting the stability of the nanomaterials in the cell medium.

On the other hand, the upright and reverse exposure apparatus is polydimethylsiloxane (poly (dimethylsiloxane), PDMS), polymethyl methacrylate (polymethylmethacrylate (PMMA), polyacrylates (polyacrylates), polycarbonates (polycarbonates), polycyclic olefins ( Polycyclic olefins, polyimides (polyimides) and polyurethanes (polyurethanes) and the like may include a microfluidic chip or wellplate exposure mechanism made of at least one polymer material selected from the group consisting of, in one embodiment of the present invention Exposure apparatus for wellplates made of transparent polycarbonates was used.

In this case, the inverted exposure apparatus is mounted so that the cultured cell layer faces the bottom, for example, the legs face upward, the top plate touches the bottom surface, and the cell layer faces the bottom.

In addition, the nanomaterials to be analyzed include gold nano, silver nano, single-walled carbon nanotube (SWCNT), multi-walled carbon nanotube (MWCNT), fullerene (fullerene, C ₆₀ ), iron nanoparticles, carbon black, titanium dioxide , At least one selected from the group consisting of aluminum oxide, cerium oxide, zinc oxide, silicon dioxide, polystyrene, dendrimer, nanoclay, and the like, and titanium dioxide was used in one embodiment of the present invention.

In addition, the dyeing may be carried out by using an absorbing dye method or a fluorescent dye method, the absorbing dye is MTT (3- (4,5-dimethyl thiazol-2-yl) -2,5-diphenyl tetrazolium bromide)), MTS (5 -(3-caroboxymethoxyphenyl) -2H-tetra-zolium inner salt), WST (4- [3- (4-Iodophenyl) -2 (4-nitrophenyl) -2H-5-tetrazolio] 1,3-benzene disulfonate) and It may be performed using a light absorbing dye selected from the group consisting of trypanblue, the fluorescent dye may be performed using an organic fluorescent dye, inorganic nanoparticles or fluorescent protein. It can also be stained in a label-free manner.

In particular, the analysis of the cell layer is preferably carried out by cell image analysis or flow cytometry methods. More preferably, it is preferable to perform cell image analysis (Image Cytometry) on the cell layer exposed and stained with the nanomaterial.

At this time, the cell image analysis may use fluorescence and absorption method including MTT analysis, in one embodiment, the cell image analysis is a bright field (formazan) formed in the cell by the MTT analysis (formazan) ( bright field) images are obtained and analyzed.

On the other hand, in the case of using flow cytometry, it is desirable to evaluate the toxicity of the nanoparticles directly through a method of distinguishing the uptake of the nanoparticles using a scattered light change and a "combination" of the fluorescence signal by modifying the conventional method.

That is, the flow cytometry results are scattered light data and fluorescence signal data, wherein the scattered light data is used to distinguish cells that have taken up the nanomaterial, and the fluorescence signal data is used to identify the nanomaterial. It is used to assess the degree of apoptosis on the cells that have taken up.

As described above, the present invention is not only modified to be easily used by the user by using the reverse exposure mechanism, but also can reduce errors due to aggregation or precipitation of nanomaterials. Furthermore, since it can be combined with in vitro cell analysis of various nanoparticles using MTT analysis, cell-specific image analysis, flow cytometry, etc., it can be widely applied to various cell effect evaluation methods currently used.

The present invention solves various problems and errors that may occur in using the protocol of the evaluation method used in the conventional chemical risk assessment in risk assessment of nanomaterials. Thus, a nanomaterial risk assessment platform based on the method of the invention can be constructed, in particular, in The risk assessment of nanomaterials in vitro may enable more accurate and reproducible assessments.

1 is a schematic diagram illustrating various cell transfer processes expected when nanoparticles are exposed to blood vessels.
2 is a diagram summarizing the factors affecting the MTT assay results.
3 is a graph showing that MTT formazan is formed only by using titanium dioxide nanomaterial.
4 is a diagram of a reversed-phase nanomaterial exposure apparatus.
5 is a schematic diagram of irradiating UV-A after exposing nanomaterials to the upright and inverted nanomaterial exposure apparatuses.
6 is a result of absorbance analysis using a microplate reader.
7 is a cell distribution chart for integrated density by analyzing absorbance of each cell obtained through image analysis.
8 is a diagram of a novel protocol of the present invention for a method for analyzing apoptosis using absorbance for nanoparticles.
Figure 9 is a graph of the survival rate of the cell, according to the concentration of titanium dioxide nanoparticles exposed to the cells of the upright and inverted nanomaterial exposure apparatus.
FIG. 10 is a diagram illustrating apoptosis reaction through microplate reader analysis and image analysis to remove the influence of nanoparticles.

Definitions of terms used in the present invention are as follows.

"Nano materials" refers to nanoparticles and nanostructured materials in which the size of the particles is less than 100 nm in dimension dimension of at least one of the three dimensions. In the present invention, in particular, the nanomaterial is used in combination with nanoparticles.

"Nanoparticles" refers to particles having a diameter in the range of 1-100 nm.

"Nanostructured material" refers to agglomerated materials or nanoparticles of a structure comprising nanosized particles.

"Nano-material hazard" is a term used to collectively refer to the dangers, risks, hazards, and toxicities of nano-sized particulate or liquid substances. Strictly speaking, "hazards" are substances or behaviors that can cause obstacles. Refers to the source of a hazard, and risk is defined as the probability or likelihood of a harmful outcome to an individual or group exposed to a specific concentration or dose of a hazardous substance. The OECD defines risk as `` Risk = Hazard × Exposure ''.

In general, the smaller the particles, the wider the specific surface area, the increased the responsiveness to the biological tissue and at the same time the toxicity is also a problem. In particular, some nanomaterials that can penetrate animal cells may pass through cell membranes or cerebrovascular membranes, which can affect unintended cells or tissues. Therefore, it is required to prepare safety management guidelines by studying the risks of these nanomaterials. According to the literature report, the risk of nanomaterials is examined in the order of carbons, silicas, TiO2, silver nanoparticles and the like.

"Tissue or cell sample" means a collection of similar cells obtained from a tissue of a subject or patient. Sources of tissue or cell samples may include solid tissue from fresh, frozen and / or preserved organ or tissue samples or biopsies or aspirates; Blood or any blood component; Cells at any time of pregnancy or development in the subject. Tissue samples may also be primary or cultured cells or cell lines.

"Apoptopsis" is used in a broad sense and is generally accompanied by one or more characteristic cellular alterations, including cytoplasmic compression, loss of plasma membrane microvilli, splitting of the nucleus, degradation of chromosomal DNA or loss of mitochondrial function. Cell death according to or regulated by mammalian rules.

Throughout this specification, the terms “comprises” and “comprising”, unless otherwise indicated in the context, include a given step or element, or group of steps or elements, but any other step or element, or step or element It should be understood that this group is not excluded.

All technical terms used in the present invention, unless defined otherwise, are used in the meaning as commonly understood by those skilled in the art in the related field of the present invention. Also, preferred methods or samples are described in this specification, but similar or equivalent ones are also included in the scope of the present invention.

Hereinafter, the present invention will be described in detail.

In vitro cell-based toxicity evaluation of nanomaterials in use is based on spectroscopy using various fluorescence and absorbing dyes to determine the extent of cell growth or death after exposure to nanomaterials in the culture of cells on the bottom of a petri dish or multiwell plate. By measuring the cytotoxicity of the nanomaterials using analytical methods. However, when measuring changes of cells exposed to nanoparticles using a microplate reader or flow cytometer, there are the following problems.

(1) The nonuniformity of the nanoparticles in the medium due to the aggregation and precipitation of the nanoparticles lowers the accuracy of the evaluation.

When the nanoparticles widely used industrially are dispersed in a cell medium (eg, RPMI-1640, DMEM), the nanoparticles are mostly aggregated due to the high ionic strength of the cell medium and precipitation occurs. Toxicity results obtained when the prepared nanoparticles were cultured on the bottom of the wellplate and evaluated for cytotoxicity by the existing in vitro-based evaluation method of exposing the toxic substances. It can be an evaluation result.

The cytotoxicity caused by the particles precipitated after aggregation and the cytotoxicity induced by the actual nanoparticles may be different in virulence mechanisms, and the higher concentration of the nanoparticles is distributed toward the bottom of the wellplate than the toxicity by the nanoparticles. Since the heterogeneity is likely to overestimate the toxicity of the actual nanoparticles, these sources of error should be clearly distinguished.

In addition, in the state in which aggregation and precipitation occurs, the nanoparticles are distributed in a non-homogeneous state in the solution phase, so that there is an excessive concentration of nanoparticles around the cells compared to the concentration of the nanoparticles administered. This overestimation of nanoparticle toxicity is also one of the major errors in the nanoparticle risk assessment system currently in use.

(2) Errors occur due to optical or catalytic properties of nanoparticles.

When apoptosis was assessed by a general spectroscopic method in the precipitated nanoparticles, false signals due to absorption, luminescence, and scattering of the precipitated nanoparticles may cause false cytotoxic results.

An example of this error is the MTT assay. The MTT assay is a test that takes advantage of mitochondria's ability to reduce the yellow water-soluble substrate MTT tetrazolium to a blue-violet, water-insoluble MTT formazan by dehydrogenase activity, and is widely used to test the toxicity of various chemicals. This technique is widely used as a method of measuring cell activity because a reproducible optical concentration can be obtained with minimal physical processing. However, if the extracellular non-biological factors influence the reduction of tetrazolium salts, fatal errors can occur in the assay results.

For example, changes in the absorbance measurements may occur depending on the pH of the cell culture and changes in adducts (serum, cholesterol, ascorbate) (Marquis et al, Analyst, 2009, 134, 425-439). In addition, recent studies on nanomaterials have reported that cytotoxic staining, such as MTT, and SWCNTs and other carbon-based materials can lead to errors in apoptosis measurement. It has recently been reported that the interaction of MTT with SWCNTs is caused by the reduced MTT formazan attached to the surface of SWCNTs and not dissolved by the solvent (Worle-Knirsch et al, Nano Lett 2006, 6, 1028-33). ) And Laaksonen et al. The oxidation reaction on the surface of PSi nanoparticles is responsible for the reduction of MTT, and can directly reduce MTT. Therefore, conducting toxicity tests on compounds of PSi particle components using the MTT screening method has limitations in obtaining quantitative results. (Laaksonen et al. Chem. Res. Toxicol. (2007)) Most of the errors of the existing MTT method are mainly due to the interaction of MTT with MTT-formazan due to non-biological factors existing outside the cell. .

Indeed, Tetrazolium-based colorimetric (MTT) assays have been widely used in a variety of biological tests, such as drug efficacy and toxicity fractions, to analyze the viability of cells using microplate reader devices. However, this method is based on the measurement of the absorbance of the entire sample, so the various errors mentioned above can occur.

In order to solve the above problems, in the present invention, "modified tetrazolium-based colorimetric (MTT) analysis and image processing" in a multiwell plate using an "inverted nanomaterial exposure apparatus" is applied to the surface dead cells ( By imaging the progress of the cell death process by apoptosis and necrosis, each cell can be analyzed to eliminate the errors that might be associated with the existing MTT method. This makes it possible to provide various information that cannot be presented by existing analysis methods or devices through more discriminating result interpretation.

In one aspect, the present invention can selectively evaluate the direct toxicity effect of nanomaterials on in vitro cells by minimizing the effect of heterogeneity of aqueous phase nanoparticles due to aggregation and precipitation using upright and inverted exposure mechanisms. It relates to cell-based assays.

In other words, the nanomaterial is exposed to the cells by using not only a normal exposure mechanism but also an "inverted exposure mechanism".

When nanoparticles cause aggregation and precipitation in the solution phase, for example, nanoparticles having an aqueous phase size of 200 nm or more occur due to aggregation and precipitation in the solution and thus the nanoparticles are distributed in a very heterogeneous state. Compared to the concentration of nanoparticles, there is an excessive concentration of nanoparticles around the cells at the bottom of the well plate.

1 is a schematic diagram illustrating various cell transfer processes expected when nanoparticles are exposed in vivo, particularly in blood vessels.

In the case of general chemicals, intracellular delivery by diffusion is the main process because there is no aggregation or precipitation phenomenon, but in the case of nanomaterials where aggregation or precipitation occurs, the heterogeneity of concentration of nanomaterials due to precipitation and passive intracellular delivery are nanomaterials. It is a significant source of error in toxicity assessment. In addition, compared to the conditions in organs such as blood vessels in vivo, cells are three-dimensionally distributed in the top, bottom, left and right, so that the heterogeneous nanoparticles may be distorted when the non-homogeneous nanoparticles are applied to a conventional method considering only cells on the bottom surface. .

Therefore, as shown in FIG. 1, in the general in vitro test, the cell is a major uptake process in which the passive particle is introduced into the cell by precipitation of aggregated hundreds of nano or micron-sized particles. However, in fact, the intrinsic toxicity of nanoparticles is a mechanism of nanoparticle toxicity in which nanoparticles in a well-dispersed state are approached to animal cells by a diffusion process, and active intracellular introduction of these nanoparticles is more important. Therefore, in many cases (manufacture) the toxicity of the nanoparticles by passive particle introduction into the cell by the precipitation of aggregated hundreds of nano or micron-sized particles in the toxicity evaluation of the nanomaterial (manufacture) They show screening effects and errors that result in too much toxicity evaluation than actual nanoparticle toxicity.

In addition, since the cells are not only present on the bottom surface but are evenly distributed in the upper and lower left and right in vivo in vivo organs, when the inhomogeneous nanoparticles are exposed in vivo, the cells on the upper and left and right sides and the cells present on the bottom surface are exposed. Will also produce errors that indicate different toxicity results. Therefore, if the toxicity evaluation of heterogeneous nanoparticles in which aggregation and precipitation occur using the conventional in vitro toxicity evaluation method, the distorted toxicity results can be obtained.

In order to solve this problem, the present invention is characterized by a three-dimensional in vitro toxicity evaluation method in which cells are placed on the upper and lower surfaces to expose heterogeneous nanoparticles.

When nanoparticle toxicity evaluation is performed by placing cells on the upper and lower surfaces, the actual nanoparticles can be compared with the cytotoxicity or toxicity mechanisms of upper and lower surfaces according to the inhomogeneity of the nanoparticles, or the nanoparticles are removed from the concentration gradient of nanoparticles caused by precipitation. Only the toxicity of the particles can be distinguished and evaluated.

In other words, the use of the upright and inverted exposure apparatus includes concentration correction due to inhomogeneity in the cell medium of the nanomaterial. Can be used to correct the concentration of exposed nanomaterials.

In addition, in the present invention, in order to solve the error problem when measuring the cell activity due to the optical or catalytic properties of the nanoparticles, it is possible to further introduce and use the cell-specific image analysis or flow cytometry. In particular, it is more preferable to use the cell-specific image analysis (Image Cytometry) together.

As shown in FIGS. 2 and 3, an error signal may be generated due to the optical or catalytic properties of the nanoparticles.

2 is a diagram summarizing the factors affecting the MTT assay results. There are three major factors: formazan formation by the reaction of MTT reagent and cells, MTT formazan formation by nanoparticles, and scattering of light by nanoparticles. It is an object of the present invention to obtain only a result value by the cells and to measure cell death.

3 is a diagram for an experiment that demonstrates the formation of MTT formazan with only titanium dioxide nanomaterial. When a total of seven samples from 0 to 6 were prepared and tested, (a) shows purple formazanes formed by MTT reagents for each sample, and absorbance according to them was shown. Changes appear purple. Finally, (c) shows the absorption spectrum of the ultraviolet / visible spectrophotometer according to the sample.

As such, errors may occur in the nanomaterial risk assessment due to the optical or catalytic properties of the nanoparticles.

Exposure of the nanoparticles in the above-mentioned inverted nanomaterial exposure apparatus can eliminate the effects of precipitated nanoparticles, but errors due to the optical or catalytic properties of the nanoparticles caused by these extracellular nanoparticles cannot be eliminated. Therefore, it is more desirable to remove errors due to the optical or catalytic properties of these nanoparticles for more accurate evaluation.

Therefore, in the present invention, in addition to the use of the inverted nanoparticle exposure apparatus, it is preferable to use cell-specific image analysis or flow cytometry to remove the interference caused by the catalytic properties of the nanoparticles.

Unlike conventional methods for measuring absorbance by including all interferences of nanoparticles, the cell-specific image analysis method measures only the absorbance of each cell. Can remove the interference.

And, in the case of using flow cytometry, flow cells somewhat modified as follows Preference is given to using the assay.

First, the forward scattered light (FS) and the side scattered light (SS) are analyzed to distinguish the cells to which the nanomaterials (nanoparticles) are attached. The front scattered light and the side scattered light in a range of one type of cell are absorbed by the nanoparticles (uptake) in the cell, so the light is refracted a lot, so the side scattered light (Side scatter (SS)) increases Separation of adherent and non-attached cells is possible. Then, only the cells to which the nanoparticles (NPs) are attached are distinguished, and the fluorescence signal data for the cells is separated and analyzed.

Using this modified flow cytometry, cytotoxicity can be assessed in vitro by measuring cellular changes caused by direct factors induced by nanoparticles, except indirect factors.

Therefore,

Attaching a cell layer to the surface of the substrate;

Exposing and staining the nanomaterials in the cell layer using upright and inverted exposure apparatuses;

Performing an analysis on the cell layer; And

It can be said that the in vitro nanomaterial risk assessment method comprising the step of quantifying the degree of apoptosis by the analysis results.

In particular, the analysis of the cell layer is preferably carried out by cell image analysis or flow cytometry methods. More preferably, it is preferable to perform cell image analysis (Image Cytometry) on the cell layer exposed and stained with the nanomaterial.

That is, as a specific example of this invention, it contains the following in A method and system for risk assessment of nanomaterials in vitro :

Attaching a cell layer to the surface of the substrate;

Exposing and staining the nanomaterials in the cell layer using upright and inverted exposure apparatuses;

Performing image cytometry for each of the cell layers; And

Quantifying the degree of apoptosis by the assay result.

In addition, the present invention is another specific example, including, in A method and system for risk assessment of nanomaterials in vitro :

Attaching a cell layer to the surface of the substrate;

Exposing and staining the nanomaterials in the cell layer using upright and inverted exposure apparatuses;

Performing flow cytometry on the cell layer; And

Quantifying the degree of apoptosis by the assay result.

In the above methods, first, the cells to be analyzed are cultured to form a cell layer.

For example, single or multiple cell layers can be cultured and used. In this case, in order to form a single cell layer, the cells to be analyzed are cultured on a surface of a multi-well plate, a slide glass, or a microfluidic cell chip, and cells floating on the medium that do not adhere to the surface are removed by flowing a medium to remove the single cell layer. Can be formed.

At this time, the conditions and methods required for the cell culture can be used a conventional method known in the art.

In particular, when using a microfluidic cell chip, by injecting a medium and a reagent into the microfluidic cell chip, there is an advantage that can be various cell-based analysis at the same time as cell culture.

Next, the present invention induces cell death by exposing the nanomaterial to the cell layer using the upright and inverted exposure apparatus.

The nanomaterials are diffused into cells over time to have various concentrations, and the cells in the cell layer undergo apoptosis by being exposed to the nanomaterials for a predetermined time.

The process of cell death is divided into apoptosis and necrosis. In contrast to normal cell division, damaged cells caused by exposure to harmful substances change in the direction of degeneration, so that metabolic processes differ from normal cells. In other words, it is possible to check the reaction mechanism of the cell's harmful substances by examining the process of death in which the cell degenerates. If the process of cell death is classified into two types, the cell is severely damaged and edema occurs in the cell. Lysis occurs, leading to a process of cell death called 'necrosis', in which cell contents are ejected out. The death process of other cells is also called 'apoptosis', which is the initial contraction of cells, which means that cell-cell bonds are destroyed in neighboring cells. In the process of apoptosis, the cell volume becomes smaller and the lining, ribosomes, glomeruli and other cell organelles in the cytoplasm contract.

Examples of nanomaterials (nanoparticles) that are concerned about risks include the following materials listed in Table 1, in which one embodiment of the present invention uses titanium dioxide nanoparticles.

In order to understand and understand the risks (toxicity) of exposure to these nanomaterials, it is necessary to understand the physical and chemical properties and physical behavior of nanomaterials, and to understand the cellular physiological in vivo.

In the present invention, the exposure of the cell layer to the nanomaterial is characterized in that it is carried out using a normal and inverted exposure apparatus.

The upright and inverted exposure apparatus is not particularly limited as long as it can expose the upper and lower surfaces of the cell layer evenly in exposing the cell layer to nanomaterials.

For example, polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polyacrylates, polycarbonates, polycyclic olefins, polyimides Exposure mechanisms for microfluidic chips or wellplates made of polymer materials such as polyimides and polyurethanes may be used. In particular, a method of manufacturing a wellplate upright and reverse exposure apparatus is shown schematically in FIG. 4.

The nanomaterials are exposed on the upper and lower surfaces of the cell layer cultured on the substrate by using the upright and reverse exposure apparatus for exposing the nanomaterials. At this time, the inverted exposure apparatus is mounted so that the cultured cell layer faces the bottom, for example, the legs of the exposure apparatus face upward, the top plate is placed on the bottom surface, and then the cell attachment surface (cell layer) of the cell culture substrate. It can be mounted facing this floor.

The use of such upright and inverted exposure devices preferably includes concentration correction due to heterogeneity in the cell medium of the nanomaterial.

That is, it is more preferable to perform concentration correction due to inhomogeneity in the cell medium of the nanomaterials while exposing the nanomaterials to the cell layer using the upright and inverted exposure apparatus. At this time, the concentration of the exposed nanomaterials can be corrected by modifying and using known formulas (theoretical and experimental formulas) that reflect the stability of the nanomaterials in the cell medium.

Known formulas (theoretical, empirical formula) and the like used in the concentration correction, reflecting the stability of the nanomaterial can be used by appropriately modifying methods known in the art in consideration of physicochemical properties such as the weight, density of the nanomaterial. For example, Justin G. Teeguarden et al . Concentration correction due to inhomogeneity of nanomaterials in cell media with reference to known papers such as "Particokinetics In Vitro: Dosimetry Considerations for In Vitro Nanoparticle Toxicity Assessments" [TOXICOLOGICAL SCIENCES 95 (2), 300-312 (2007)]. Can be performed.

In this way, the cell layer is placed on the upper and lower surfaces using the upright and inverted exposure apparatus to expose the heterogeneous nanoparticles in three dimensions, thereby comparing the upper and lower cytotoxicity or toxic mechanisms according to the degree of inhomogeneity of the nanoparticles or by generating the nanoparticles. Eliminate toxicity due to the concentration gradient of the particles.

Therefore, in another aspect, the present invention may include a method for measuring heterogeneous concentrations of nanomaterials according to colloidal properties, such as the degree of aggregation and precipitation of the nanoparticles, using upright and inverted exposure mechanisms.

Next, the cell layer exposed to the nanomaterial is stained using the upright and inverted exposure mechanisms.

Absorption staining or fluorescence staining may be used. In one embodiment of the present invention, the light absorbing staining method is used.

Absorption dyes used for absorbing dye include MTT (3- (4,5-dimethyl thiazol-2-yl) -2,5-diphenyl tetrazolium bromide)) and MTS (5- (3-caroboxymethoxyphenyl) -2H-tetra- zolium inner salt), WST (4- [3- (4-Iodophenyl) -2 (4-nitrophenyl) -2H-5-tetrazolio] 1,3-benzene disulfonate) and trypanblue Can be used. In addition, fluorescent dyes used for fluorescent dyeing include organic fluorescent dyes such as DCF, DiOC6, Hoechest 33432; Or inorganic nanoparticles such as quantum dots (QD); Or fluorescent proteins such as GFP, RFP, and YFP.

At this time, in the case of using absorbance staining, thereafter cell image analysis (Image Cytometry); In the case of using fluorescent staining, flow cytometry is generally used.

In addition, the dyeing may be done in a label-free manner.

As described above, the present invention may further perform cell-specific image analysis or flow cytometry on the cell layer in which the nanomaterials are exposed and stained through the upright and inverted exposure mechanisms, and the degree of apoptosis is determined by the analysis result. Can be quantified.

For example, cell-specific image analysis may be performed using fluorescence and absorption methods including MTT analysis.

That is, by applying modified MTT (Tetrazolium-based colorimetric) analysis and image processing in a multiwell plate using an inverted nanomaterial exposure apparatus, apoptosis and necrosis of cells adhered to the surface Analyze the progress of the cell death process.

The MTT assay is a water-soluble MTT formazan (3- (4,5-dimethylthiazol-2-yl) -2,5 which is blue-purple to MTT tetrazolium, a yellow water-soluble substrate by dehydrogenase action. As a test to measure the activity of mitochondria in cells reduced to -diphenyl-tetrazolium bromide, it is widely used to test the toxicity of various chemicals.

At this time, the imaging of the cell layer is a light source selected from the group consisting of a tungsten lamp (tungsten lamp), LED light source and laser light source as a light source in the visible light region for optical image acquisition; CCD (Charge Coupled Device) camera for detecting the collected cell image from the objective lens: a cell culture platform for placing a cell culture system comprising a single cell layer; A device for selectively passing a wavelength at a light source to obtain an image in the maximum absorption region applied to the absorbing dye used, and analyzing the image obtained from the CCD camera to the cell-specific absorbance factor, number of cells and shape factor values. One or more apoptosis factors selected from the group consisting of can be carried out using a cell image analysis device comprising an image processing program that can quantify the cell death process.

In particular, when MTT is used as the light absorbing dye, a wavelength selectively passed to obtain an image in the maximum light absorbing region is a wavelength region of 550 nm ± 60 nm, and when MTS formazan is used as the light absorbing dye, an image in the maximum light absorbing region is obtained. For example, the wavelength selectively passed is 490 nm ± 60 nm, and when trypan blue is used as the light absorbing dye, the wavelength selectively passed to obtain an image in the maximum absorption range is preferably 590 nm ± 80 nm. In addition, when MTT is used as the light absorbing dye, it is preferable to give an optimal time of 10 to 240 minutes to induce formation of formazan crystals by oxidation / reduction reaction. When trypan blue is used, the image can be analyzed by giving an optimal time to dyeing with the exposed dye within 3 minutes.

The quantification step of the degree of apoptosis is the mean absorbance and the cell-specific mean absorbance and cell-specific factors as a form factor value or an absorbance factor selected from the group consisting of area and circularity occupied by each cell from the photographed image. One or more cell-specific absorbances selected from the group consisting of total absorbance can be analyzed and quantified.

Circularity = 4π x (cell area / cell circumference ² )

Mean Absorabnce by Cell = Mean Absorbance of Each Pixel in Cell Area

Integrated Absorbance per cell = average absorbance per cell x number of pixels in the area occupied by the cell

In the step of quantifying the degree of apoptosis, a single-variable analysis of variables including the absorbance of each cell obtained through image analysis can be performed to obtain a dose-response curve and to quantify the progress of cell death. Regarding the analysis using the absorbance image analysis for each cell, reference may be made to Korean Patent Application No. 10-2009-0133151.

In another method, the present invention may collect scattered light data and fluorescence signal data through flow cytometry to analyze the degree of apoptosis by measuring the incidence of reactive oxygen species.

In particular, as described above, it is characterized by using the following modified flow cytometry method.

-Forward Scatter (FS) and Side Scatter (SS) To distinguish cells with attached nanoparticles (NPs). The front scattered light and the side scattered light in a range of one type of cell are absorbed by the nanoparticles (uptake) in the cell, so the light is refracted a lot, so the side scattered light (Side scatter (SS)) increases Separation of adherent and non-attached cells is possible.

The cell change of the nanoparticles is measured and analyzed only by combining the scattered light data analysis result and the fluorescence signal.

In quantifying the flow cytometry results, intracellular nanoparticles are linked by forward scattering (FS) and fluorescence emission signals of cells whose lateral scattering (SS) intensity is measured at a certain ratio or more relative to control cells. Cell death induced by the introduction of and various mechanism changes are measured and based on this, the nanoparticle dose-response relationship is to objectively assess the direct harmfulness of nanoparticles.

At this time, the flow cytometry apparatus that can be used may use a commercially available flow cytometer (Flow Cytometry or Fluorescence Activated Cell Sorter), or may use a microfluidic flow cytometry.

In particular, the microfluidic flow cytometry instrument is one example.

Light sources selected from the group consisting of tungsten lamps, mercury and halogen lamps, LED light sources, laser light sources, and the like as light sources in the ultraviolet and visible light region;

A detection device comprising a photomultiplier tube (PMT) or a charge coupled device (CCD) for detecting an optical signal scattered or fluoresced from a cell; A cell culture platform for placing a cell culture system comprising a cell layer;

A device for selectively passing wavelengths in the light source to obtain an image in the maximum absorption or fluorescent region applied to the fluorescent dye used;

Analyze the data obtained from the device to extract one or more apoptosis factors selected from the group consisting of cell forward (FS) and lateral (SS) scattering intensity, fluorescence intensity, number of cells and shape factor values, and apoptosis process It can include data processing programs that can be quantified. Regarding such microfluidic flow cytometry apparatus, reference may be made to Korean Patent Application No. 10-2009-0076436.

The method of the present invention is useful in securing safety guidelines for harmful effects on the production, environment, and health of nanomaterials by making it possible to accurately grasp and understand the dangers (toxicities) of exposure to nanomaterials.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example  1: Formulation Normal ) And Upright ( Inverted ) Nanomaterial  Manufacture of exposure device

As shown in FIG. 4, a transparent poly carbonate and an acrylic bond were prepared.

A 1.5 cm x 1.5 cm x 0.2 cm square top plate was attached to two 1.5 cm x 0.8 cm x 0.3 cm rectangles to form a desk-shaped structure. As a cell culture substrate, a poly styrene plate having a size of 1.5 cm x 1.5 cm was used. The nanomaterial exposure apparatus and the cell culture substrate were washed with soapy water and soaked in 70% ethanol for 30 minutes.

Example  2: cell culture and cell layer formation

Plasma-treated 1.5 cm x 1.5 cm square polystyrene plates were placed in a 12-multiwell plate and the nanomaterial exposure apparatus was fixed on the edges of the polystyrene plates.

Cancer cell lines (Hela cell line, Korea Biological Resource Center, Korea) cultured in cell culture vessels were extracted into suspensions having 10 ⁴ to 10 ⁵ cells / mL from the bottom surface and injected into multiwell plates. The injected cells formed a single layer of adherent cell population on the surface of the polystyrene plate.

At this time, Dulcecoo's modified Eagle's medium (DMEM, Gibco, Grand Island, NY, USA, with 10% fetal bovine serum (Gibco, Grand Island, NY, USA)), 1% Penicillin-Streptomcyin (Gibco, Grand Island, NY, USA))) medium was used and incubated for about 24 hours.

Example  3: formulation and Upright  Using the exposure device Nanomaterial  process

The cell culture substrates to which the animal cells are attached to the upright and inverted nanomaterial exposure apparatuses were fixed up and down respectively.

Sizing nanomaterial exposure removed the medium while the cells were incubated. The inverted nanomaterial exposure was mounted so that the legs of the nanomaterial exposure apparatus were facing upward, the top plate was placed on the bottom surface, and the cell attachment surface of the cell culture substrate was facing the bottom.

Then, the nanomaterial exposure apparatus in which the cell layer was fixed was installed in a multiwell plate and the nanomaterial was exposed. That is, the cell culture solution containing the nano-material (TiO ₂ ) was exposed, at which time the solution was put in a volume (3ml) so as to be locked to the cell culture substrate from the inverted nanomaterial exposure apparatus.

Upright nanomaterial exposure exposed UV-A for 1 hour and 20 minutes at the bottom of the multi well plate, and reverse nanoparticle exposure exposed UV-A for 1 hour and 20 minutes at the top of the multi well plate (FIG. 5).

After the UV-A exposure, the plate was placed in an incubator at 37 ° C. and 5% carbon dioxide concentration, and exposed to nanoparticles (TiO ₂ ) for a total of 20 hours, including UV-A exposure time.

Comparative example  1: Comparison of Cellular Image Analysis and Conventional Microplate Readers

Microplate Reader is a device used for qualitative and quantitative analysis of reacted materials in Microplate Well. Absorption, emission, fluorescence, etc It is used in the field of cell physiology, molecular biology, and immunology, and light from light sources is irradiated through 96 or more paths to samples made of light of specific wavelengths through various filters and monochromators. Fluorescent or transmitted light generated according to the composition and concentration of the sample contained in the well of the sample is analyzed by the detector. Based on the results measured in this way, the proliferation and cell viability of cells can be observed.

First, the inventors looked at the results of analyzing the degree of cell death with the plate reader. In the case of using a microplate reader, the overall absorbance is analyzed.

The results are shown in FIG.

The scattered graphs of the cell area and the resulting absorbance of the nanoparticles, dead cells and surviving cells are shown in the scatter graph. It can be seen that the absorbance is generated by the nanoparticles even if they are not alive. It can be seen that this is a factor that can cause an error in how to view the cell death process using.

Next, the degree of apoptosis was analyzed by using cell cytometry. In other words, the absorbance of each cell was analyzed through image analysis to examine the distribution of cells in integrated density.

And the result is shown in FIG.

It is a figure which shows the death on the left side and the survival on the right side centering on the baseline (broken line) which judges the criterion of death. As a result, when the killing process was analyzed only for cells excluding the nanoparticles, as the concentration of the nanoparticles increases, the degree of death increases. This can be seen through.

In other words, in the cell-specific image analysis, unlike the conventional method of measuring absorbance including all interferences of nanoparticles, the absorbance of each cell is measured by selectively measuring only the result of the interaction between the nanoparticles and the cells. As a result, the interference of nanoparticles due to spectroscopic characteristics can be eliminated.

Example  4. Apoptosis  analysis( Nanomaterial  To evaluate)

Existing MTT assays were performed with modified MTT assays and controls according to the method of the present invention. The process of the existing method and the modified method presented in the present invention are shown in FIG. 8, respectively.

In order to perform the conventional MTT assay, cells were cultured in an appropriate amount in 96 wells, and 100 μL of a non-aqueous yellow MTT solution diluted 10-fold in medium was added to each well after treatment with toxic substances. Store in an incubator at 37 ° C and 5% carbon dioxide concentration to produce intracellular formazan crystals, remove excess media after about 4 hours, and 200 μL of DMSO in each well to dissolve the insoluble formazan formed in the cells. After addition the absorbance was analyzed at 595 nm using a microplate reader.

And MTT analysis was performed according to the method of the present invention.

After 20 hours of nanomaterial treatment, cells were appropriately cultured in multi well plates, and nanomaterials were removed, and 1.5 ml of MTT solution diluted 10-fold with medium was exposed. Since the absorbance values of MTT formazan crystals should be within the range of effective pixel values in bright field images, the formation time of formazan crystals was given for 30 minutes in order to optimize the crystal generation time.

Unlike the conventional MTT retrieval method, it is not necessary to dissolve the crystals of formazan with an organic solvent (for example, DMSO), and the bright field remains in the formazan cell by using a microscope for image acquisition and processing. field) images were acquired.

Because of the maximum absorption wavelength at about 550 nm of MTT formazan, a bandpass filter having a maximum transmittance at 45 nm and a band width of 45 nm was used to obtain a short wavelength light source when obtaining an optical image.

Cell viability was measured according to the above method.

First, the cell survival rate according to the concentration of the titanium dioxide nanoparticles exposed to the cells of the upright and inverted nanomaterial exposure apparatus in Example 3 was also confirmed and shown in FIG. 9.

FIG. 9 (a) is a graph showing experiments on titanium dioxide nanoparticles having an aqueous solution size of 300 nm on average, and FIG. 9 (b) is graph of titanium dioxide nanoparticles having an aqueous solution size of 70 nm on average. It is a figure which carried out experiment.

In the results of (a), when the large particles were precipitated, the cytotoxicity of the upright and inverted particles was different. That is, it can be seen that the use of the inverted exposure device is significant. On the other hand, in (b), precipitation did not occur much, and there was no significant difference in the cytotoxicity between the upright and inverted particles.

In other words, it can be seen that the error according to the cytotoxicity when the precipitation of nanomaterials occurs can be significantly reduced by the method of the present invention using the formulation and the reverse exposure apparatus.

However, even if MTT formazan does not occur, an error in which absorbance occurs due to scattering of the nanoparticles itself was found (FIG. 9 (a)).

Therefore, apoptosis was measured by using absorbance through image analysis to remove the effect of the nanoparticles.

That is, to recognize the comparative example 1 and the error that there are many problems in observing apoptosis reaction to the nanoparticles using a microplate reader, and to remove the spectroscopic effects of the nanoparticles through image analysis.

10 is a diagram showing that an error occurs in the absorbance measurement by the nanoparticles. (a) shows the result of apoptosis obtained from image analysis and apoptosis result in the upright nanomaterial exposure apparatus, and (b) shows the result of apoptosis and apoptosis result obtained from image analysis. The result is a comparison.

That is, as shown in Figure 10 it can be seen that the analysis through the image analysis is different from the analysis using a microplate reader.

Therefore, in combination with the above results, conventional cell death assays caused by scattering of nanoparticles themselves, i.e., by optical or catalytic reactivity of nanoparticles, by using an inverted nanomaterial exposure apparatus and further analyzing image analysis results It can be seen that the error can be reduced.

In other words, the results are applied to the apoptosis measurement method using the inverted nanomaterial exposure method and cell image analysis technique to quantify the degree of cell death to remove various causes of measurement error caused by the intrinsic physicochemical properties of the nanomaterial. In addition, it shows that only the response of cells generated by the interaction between nanoparticles and cells can be selectively measured and analyzed.

Claims (18)

Attaching a cell layer to the surface of the substrate;
Exposing and staining the nanomaterials in the cell layer using upright and inverted exposure apparatuses;
Performing an analysis on the cell layer; And
, In vitro nanomaterials risk assessment comprises the step of quantifying the degree of cell death by the analysis result.
The method of claim 1, wherein the cell layer is formed on a microfluidic cell chip, a multi-well or a slide glass.
The method of claim 1, wherein the cell layer is a single or multiple cell layer.
The method of claim 1, wherein using the upright and inverted exposure apparatus comprises concentration correction due to heterogeneity in the cell medium of the nanomaterial.
According to claim 1, wherein the upright and inverted exposure apparatus is polydimethylsiloxane (poly (dimethylsiloxane), PDMS), polymethyl methacrylate (polymethylmethacrylate (PMMA), polyacrylates (polyacrylates), polycarbonates, And a microfluidic chip or wellplate exposure apparatus made of at least one polymer material selected from the group consisting of polycyclic olefins, polyimides, and polyurethanes.
The method of claim 1 wherein the upright and inverted exposure apparatus comprises an exposure mechanism for a microfluidic chip or wellplate.
The method of claim 1, wherein the inverted exposure device is mounted so that the cultured cell layer faces the bottom, and is exposed to the nanomaterial, and the method is used to correct the concentration of the exposed nanomaterial.
The method of claim 1, wherein the nanomaterial is gold nano, silver nano, single-walled carbon nanotube (SWCNT), multi-walled carbon nanotube (MWCNT), fullerene (fullerene, C ₆₀ ), iron nanoparticles, carbon black, titanium dioxide And at least one nanomaterial selected from the group consisting of aluminum oxide, cerium oxide, zinc oxide, silicon dioxide, polystyrene, dendrimer, and nanoclays.
The method as claimed in claim 1, wherein the dyeing is carried out by using an absorption dyeing method or a fluorescent dyeing method.
The method of claim 1 wherein the dyeing is in a label-free manner.
The method of claim 9, wherein the absorbance dye is MTT (3- (4,5-dimethyl thiazol-2-yl) -2,5-diphenyl tetrazolium bromide)), MTS (5- (3-caroboxymethoxyphenyl) -2H-tetra -zolium inner salt), WST (4- [3- (4-Iodophenyl) -2 (4-nitrophenyl) -2H-5-tetrazolio] 1,3-benzene disulfonate) and trypanblue The method characterized in that it is carried out using the absorbing dye selected.
The method of claim 9, wherein the fluorescent dye is performed using an organic fluorescent dye, an inorganic nanoparticle, or a fluorescent protein.
The method of claim 1, wherein the analysis of the cell layer is performed by cell image analysis or flow cytometry.
The method of claim 13, wherein the analysis of the cell layer is performed by a cell image analysis method.
15. The method of claim 14, wherein said cell image analysis uses fluorescence and absorption methods including MTT analysis.
The method of claim 15, wherein the cell image analysis is performed by obtaining a bright field image with intracellular formazan formed by MTT analysis.
The method of claim 13, wherein the flow cytometry uses scattered light data and fluorescence signal data.
18. The method of claim 17, wherein the scattered light data is used to distinguish cells uptake of the nanomaterial, and the fluorescence signal data is used to evaluate the degree of apoptosis for the cells that absorbed the nanomaterial. How to.

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