Assessing Toxicity of Nanoparticles with In Vitro Cell Based Assays

Chandrasekaran Vasudevan1, Jeffery R. Haskins1, William E. Burmeister2, Tom Komorowski2 and Lori Bestervelt2
www.cellomics.com
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Cellomics, Inc., 100 Technology Drive, Pittsburgh PA 15219 NSF International, 789 N. Dixboro Road, Ann Arbor MI 48105

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Assessing Toxicity of Nanoparticles with In Vitro Cell Based Assays
Chandrasekaran Vasudevan1, Jeffery R. Haskins1, William E. Burmeister2, Tom Komorowski2 and Lori Bestervelt2 1 Cellomics, Inc., 100 Technology Drive, Pittsburgh PA 15219 and 2NSF International, 789 N. Dixboro Road, Ann Arbor MI 48105
www.cellomics.com

Introduction
One of the many formats of cell based assays is High Content Screening (HCS) assays, where typically multiplexed targets of interest in compound treated cells are imaged and analyzed in an automated fashion. A typical HCS assay can provide data on such multiple parameters as cell density, cellular morphology and size, localization and fluorescent intensity of the targets (usually more than one) in a cell. A HCS assay is usually performed in a micro-plate with 96 or 384 wells, allowing for screening of a large number of compounds against one or multiple targets of interest. This not only allows for a more efficient use of reagents and other resources, but also provides for direct and easy cross correlation of compound effects on multiple cellular targets from the same experiment. HCS assays are especially useful in studying cytotoxicity of compounds, because it allows for multiplexing targets of relevance for cytotoxicity such as nuclear morphology, permeability, membrane potential, pH, mitochondrial transmembrane potential, apoptosis, changes in cellular concentration of Ca2+ and other ions or oxidative stress. By using an appropriate combination of fluorescent reagents, a single HCS experiment can provide valuable data for many of the above mentioned targets. These cell based HCS assays also act as a valuable first step prior to studying the toxicological effects of compounds in animal testing, a process that is much more expensive in terms of resources including time. A typical HCS experiment in a 96 well plate can provide useful data in about 3 hours post-exposure of cells to nanoparticles, depending on the nature of cellular targets selected for studying. Also, all of the steps involved in sample preparation for HCS can be automated, providing for a very efficient way to screen for a variety of targets or nanoparticles in a short amount of time. Due to their many desirable properties nanoparticles and nanomaterials are finding increasing use in a number of commercial products, including possible use as transporters for drugs to treat cancer (1, 2). However, the toxicological effects if any, of such widespread use of these materials is not well characterized. One reason for this could be the relatively short time of commercial use of these materials and the consequent lack of any large scale toxicological studies using such nanomaterials. The second more important reason could be the lack of well characterized toxicological targets and assay methods for studying the effects of nanomaterials on these targets. Only recently has a working group report been published that details screening strategy for various biological targets that can be affected by nanomaterials (3). There are also several recent reports that describe efforts to study the toxicity mechanisms of nanoparticles in various biological systems (4-7). Using a HCS approach we have studied the toxicity of nanotubes in two human tissue derived cell lines: (i) A549, a lung cancer derived cell line and (ii) HepG2, a liver carcinoma derived cell line. The targets/biological indicators of toxicity we chose include cell membrane permeability, nuclear morphology, mitochondrial transmembrane potential and induction of apoptosis. Our results show that single-walled nanotubes (SWNT) are more potent than multi-walled nanotubes (MWNT) or C60 Fullerene in affecting the mitochondrial transmembrane potential in the two cell lines studied. Interestingly none of the three different materials have any significant effect on cell membrane permeability. The cell cycle status of A549 cells treated with these nanomaterials were analyzed by measuring the total intensity of Hoechst-33342 staining in the nuclei. Our data from these experiments clearly shows that SWNT shifts the frequency distribution of nuclear staining by Hoechst-33342 towards a sub G0/G1 cell cycle status in a dose dependent manner. This observation suggests that A549 cells are undergoing apoptosis when exposed to

SWNT. Other nuclear morphological characteristics that are indicative of apoptosis, such as fragmenting and disorganized nuclei were also clearly visible in cells treated with SWNT, while absent in cells treated with C60 Fullerene. Our results are similar to what has been observed in HEK293 cells treated with SWNT (6). Currently we are looking for the presence of other markers for apoptosis in cells treated with nanoparticles, to better understand the mechanism of cytotoxicity induced by exposure to nanoparticles. Thus using an HCS approach we have started to understand some of the mechanisms of nanoparticles induced cytotoxicity. We believe that the HCS approach will provide a very efficient way by allowing toxicologists to multiplex relevant targets in a

Results
Table 1: Assay strategy for measurement of cytotoxicity of nanoparticles in cells
Target Nucleus Mode of Action DNA binding Fluorescence Emission Blue Green Cellular Measurement Nuclear area/ intensity Intensity in nuclear region

Figure 3: Mitochondrial transmembrane potential is significantly reduced in A549 cells treated with 72 g/mL of SWNT for 24 hours.

Figure 6: SWNT causes an accumulation of A549 cells in sub G0/G1 state, while C60 Fullerene does not affect cell cycle status of these cells
9000 8000 7000 6000 5000 4000 3000 2000 1000 0

Conclusions
Cytotoxicity of nanoparticles can be assayed using a cell based in vitro approach. SWNT affect mitochondrial transmembrane potential without affecting cell membrane permeability. MWNT and C60 Fullerenes have no effect on the mitochondrial transmembrane potential or cell membrane permeability at comparable concentrations. MWNT appears to affects mitochondrial transmembrane potential significantly (comparable to SWNT) at very high (10x of SWNT) concentrations. SWNT do not quench fluorescence in the blue, green and red and far-red wavelength regions. SWNT affects cell cycle status by causing an accumulation of cells in the sub G0/G1 phase. Nuclei of cells that are in the sub G0/G1 phase show morphological features that are similar to cells undergoing apoptosis. We are in the process of studying the effect of nanoparticles on mitochondria using electron microscopy. The relatively short processing times of 96 well plates, post-incubation with nanomaterials and quick scan times of these plates, makes HCS an ideal method for large scale screenings of nanoparticles for cytotoxicity. Further investigations are needed to better understand the mechanisms by which nanoparticles induce apoptosis and also to decipher other cellular pathways that are affected by nanoparticles.

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Methods & Materials
HepG2 cells (ATCC # HB-8065) growing in flasks, were trypsinized, dispersed by passing through a 26G1/2 syringe needle and plated at 8,000 cells per well into collagen I coated 96 well micro plates. Cells were allowed to attach and cultured overnight at 37O C in a 5% CO2 incubator. A549 cells (ATCC # CCL-185) growing in flasks, were trypsinized and plated at 6,000 cells per well into tissue culture treated 96 well micro-plates. Cells were allowed to attach and cultured overnight at 37O C in a 5% CO2 incubator. SWNT (Cat # 900-1300), MWNT (Cat # 900-1200) and C60 Fullerene (Cat # 600-9980) were obtained from SES Research, Houston, Texas. Stock solutions of these materials were made as a suspension in 1% Acacia Gum (AcGum, Sigma Cat # G 9752). The cells growing in 96 well plates were treated with varying doses of the nanoparticles for 24 hrs. The cells were in a 5% CO2 incubator at 37O C during this incubation. Staining with Hoechst-33342 (0.2 g/mL or 1 g/mL), Mitotracker Orange (1 M) and YOYO-1 (0.25 M) was done during the final 30 minutes of incubation with the nanoparticles. Stained cells in 96 well plates were then fixed with 3.7 % formaldehyde made up in HBSS and washed with HBSS. To determine if SWNT did cause fluorescence quenching, beads that fluoresce in the blue, green, red and far red region (Molecular Probes) were mixed with varying concentrations of SWNT in 96 well plates and imaged on the ArrayScan® HCS Reader. Fluorescence intensity was quantitated using the Target Activation BioApplication (see below). 96 well micro-plates with cells treated and stained as described above were scanned in an ArrayScan HCS Reader using the Target Activation BioApplication (see below) or the Compartmental Analysis BioApplication (see below) with a 10X, 0.3 NA or a 20X, 0.4 NA objective. Data analysis was performed after exporting data from vHCSTM: View, a data visualization tool, to Microsoft Excel. Description of Target Activation BioApplication The Target Activation BioApplication for the ArrayScan HCS Reader is an automated image analysis algorithm that can quantify fluorescent intensities for multiple targets in the nuclear region or entire cellular regions of individual cells. Additionally it can also quantitate changes in nuclear morphology and size. Description of the Compartmental Analysis BioApplication The Compartmental Analysis BioApplication for the ArrayScan HCS Reader is an automated image analysis algorithm that can quantify fluorescent intensities of multiple targets in the nuclear and other cellular regions of individual cells. The BioApplication can also measure fluorescent intensity, size and number of distinct features in the cytoplasmic region. It can also report differences and ratios of fluorescent intensities from different cellular regions. Additionally it can also quantitate changes in nuclear morphology and size.

Dye binds only Cell Membrane to nucleus of Permeability permeabilized cells Binds to Mitochondrial mitochondria transmembrane depending on transmembrane potential potential

N uc l e a r Tot a l I nt e n si t y

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Figure 1: Only SWNT cause a significant decrease in mitochondrial transmembrane potential in HepG2 Figure 4: Cell membrane permeability is not cells affected by nanoparticles
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Mitochondrial staining by MitoTracker Orange in A549 cells imaged on the ArrayScan HCS Reader with a 20X objective and running the Compartmental Analysis BioApplication. Panel A is cells treated with media only and panel B is cells exposed to 72 g/mL SWNT for 24 hours. Dark spots (yellow arrow heads) in panel B are most likely to be aggregates of SWNT.

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Frequency distribution of nuclear intensity of Hoechst 33342 (0.2 g/mL) stained A549 cells with various doses of SWNT (panel A) or C60 Fullerene (panel B). Total number of cells for each treatment ranged from about 30,000 to 55,000 cells.

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Figure 7: Nuclei of A549 cells treated with SWNT show apoptosis morphology

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References
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Effect of varying doses of different nanoparticles on the mitochondrial transmembrane potential of HepG2 cells. Cells were treated and stained as described earlier. Data is mean ± SD from 8 wells for each treatment, except for AcGum, which is mean ± SD from 16 wells. ** indicates data that was statistically significant (p < 0.05 by Student’s t test) from AcGum and media controls.

Cell membrane permeability is not affected by exposure to different nanopartilces for 24 hours. A549 cells were treated and stained as described earlier. Data is mean ± SD from 8 wells for each treatment, except for AcGum, which is mean ± SD from 16 wells. Valinomycin data was added as an indicator of membrane permeability induced by the K+ ionophore.

Nuclear morphology of A549 cells: (A) untreated, (B) C60 Fullerene 720 g/ mL and (C) SWNT 72 g/mL. Cells were stained with H-33342 (0.2 g/mL) and imaged in an ArrayScan HCS Reader with a 10X objective. Notice the altered nuclear morphology and lower cell density in panel C only.

Figure 2: Only SWNT cause a significant decrease in mitochondrial transmembrane potential in A549 cells also
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Figure 5: SWNT does not quench fluorescence in the blue, green and red wavelength region
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Figure 8: MWNT affect mitochondrial transmembrane potential at very high doses.
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Effect of varying doses of different nanoparticles on the mitochondrial transmembrane potential of A549 cells. Cells were treated and stained as described earlier. Data is mean ± SD from 8 wells for each treatment, except for AcGum, which is mean ± SD from 16 wells. ** indicates data that was statistically significant (p < 0.05 by Student’s t test) from AcGum and media controls.

Multicolored fluorescent beads in 96 well plates were mixed with various concentrations of SWNT and images on the ArrayScan HCS Reader as described earlier. Panels A (blue), C (green) & E (red) are beads with no SWNT and panels B (blue), D (green) & F (red) are beads mixed with 1780 g/mL SWNT. Panel G shows the fluorescent intensity of the beads with varying concentrations of SWNT in the red wavelength region. Similar plots were obtained for the other wavelength regions also.

A549 cells in 96 well plates were treated with SWNT or very high concentration of MWNT or C60 Fullerene for 24 hours. Cells were stained with mitochondrial potential stain and processed as described earlier. Data is mean ± SD from 16 wells (media and AcGum) or 24 wells (others). Only very high doses of MWNT had an effect on mitochondrial potential staining, while C60 Fullerene had no effect at this high dose also.

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