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
Cellomics, Inc., 100 Technology Drive, Pittsburgh PA 15219
1

NSF International, 789 N. Dixboro Road, Ann Arbor MI 48105

2

Cellomics, Inc.
100 Technology Dr.
Pittsburgh, PA 15219
US toll-free: (800) 432-4091
Main: (412) 770-2200
Facsimile: (412) 770-2450

Cellomics Europe
Wyvols Court
Swallowfield
Reading
Berks
RG7 1WY
Main: +44 118 9880 262
Facsimile: +44 118 9880 362
www.cellomics.com
Assessing Toxicity of Nanoparticles with In Vitro Cell Based Assays
Chandrasekaran Vasudevan1, Jeffery R. Haskins1, William E. Burmeister2, Tom Komorowski2 and Lori Bestervelt2
1 www.cellomics.com
Cellomics, Inc., 100 Technology Drive, Pittsburgh PA 15219 and 2NSF International, 789 N. Dixboro Road, Ann Arbor MI 48105

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
SWNT. Other nuclear morphological characteristics that are Figure 3: Mitochondrial transmembrane potential
indicative of apoptosis, such as fragmenting and disorganized Results Figure 6: SWNT causes an accumulation of A549
nuclei were also clearly visible in cells treated with SWNT, while
absent in cells treated with C60 Fullerene. Our results are similar Table 1: Assay strategy for measurement of
is significantly reduced in A549 cells treated with
72 Pg/mL of SWNT for 24 hours.
cells in sub G0/G1 state, while C60 Fullerene does Conclusions
not affect cell cycle status of these cells
to what has been observed in HEK293 cells treated with SWNT cytotoxicity of nanoparticles in cells Cytotoxicity of nanoparticles can be assayed using a cell
(6). Currently we are looking for the presence of other markers x
A based in vitro approach.
Media AcGum- 0.1% SW-72 SW-36 SW-18

for apoptosis in cells treated with nanoparticles, to better Fluorescence Cellular

9000

provide data on such multiple parameters as cell density, understand the mechanism of cytotoxicity induced by exposure Target Mode of Action
8000
x SWNT affect mitochondrial transmembrane potential
Emission Measurement
7000

cellular morphology and size, localization and fluorescent to nanoparticles.

6000 without affecting cell membrane permeability. MWNT
intensity of the targets (usually more than one) in a cell. A HCS Nuclear area/ 5000
and C60 Fullerenes have no effect on the mitochondrial
Thus using an HCS approach we have started to understand Nucleus DNA binding Blue
intensity
4000

assay is usually performed in a micro-plate with 96 or some of the mechanisms of nanoparticles induced cytotoxicity.
3000 transmembrane potential or cell membrane permeability at
384 wells, allowing for screening of a large number of Dye binds only 2000

comparable concentrations.
We believe that the HCS approach will provide a very efficient 1000

compounds against one or multiple targets of interest. This not Cell Membrane to nucleus of Intensity in MWNT appears to affects mitochondrial transmembrane
way by allowing toxicologists to multiplex relevant targets in a Green x
0

only allows for a more efficient use of reagents and other Permeability permeabilized nuclear region N uc l e a r Tot a l I nt e n si t y
potential significantly (comparable to SWNT) at very high
resources, but also provides for direct and easy cross cells (10x of SWNT) concentrations.
correlation of compound effects on multiple cellular targets
from the same experiment.
Methods & Materials Binds to
Mitochondrial mitochondria Mitochondrial staining by MitoTracker Orange in A549 cells
3000
B Media AcGum-0.1% C60-720 C60-360 C60-180 x SWNT do not quench fluorescence in the blue, green and
red and far-red wavelength regions.
Intensity in
HCS assays are especially useful in studying cytotoxicity of HepG2 cells (ATCC # HB-8065) growing in flasks, were transmembrane depending on Orange Red imaged on the ArrayScan HCS Reader with a 20X objective and
2500

x SWNT affects cell cycle status by causing an accumulation

x mitochondria
compounds, because it allows for multiplexing targets of potential transmembrane running the Compartmental Analysis BioApplication. Panel A is of cells in the sub G0/G1 phase.
2000

trypsinized, dispersed by passing through a 26G1/2 syringe

potential cells treated with media only and panel B is cells exposed to Nuclei of cells that are in the sub G0/G1 phase show
1500

relevance for cytotoxicity such as nuclear morphology, needle and plated at 8,000 cells per well into collagen I coated x

morphological features that are similar to cells undergoing

1000

permeability, membrane potential, pH, mitochondrial 96 well micro plates. Cells were allowed to attach and cultured 72 Pg/mL SWNT for 24 hours. Dark spots (yellow arrow heads)
Figure 1: Only SWNT cause a significant decrease in apoptosis.
500

transmembrane potential, apoptosis, changes in cellular overnight at 37O C in a 5% CO2 incubator. in panel B are most likely to be aggregates of SWNT.
We are in the process of studying the effect of
0

concentration of Ca2+ and other ions or oxidative stress. By x A549 cells (ATCC # CCL-185) growing in flasks, were mitochondrial transmembrane potential in HepG2 x

using an appropriate combination of fluorescent reagents, a trypsinized and plated at 6,000 cells per well into tissue culture Figure 4: Cell membrane permeability is not N uc l e a r Tot a l I nt e n si t y

nanoparticles on mitochondria using electron microscopy.

single HCS experiment can provide valuable data for many of
cells The relatively short processing times of 96 well plates,
treated 96 well micro-plates. Cells were allowed to attach and affected by nanoparticles Frequency distribution of nuclear intensity of Hoechst 33342 x

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
cultured overnight at 37O C in a 5% CO2 incubator. (0.2 Pg/mL) stained A549 cells with various doses of SWNT post-incubation with nanomaterials and quick scan times
x SWNT (Cat # 900-1300), MWNT (Cat # 900-1200) and C60 300 (panel A) or C60 Fullerene (panel B). Total number of cells for of these plates, makes HCS an ideal method for large scale
Fullerene (Cat # 600-9980) were obtained from SES Research, each treatment ranged from about 30,000 to 55,000 cells. screenings of nanoparticles for cytotoxicity.
Houston, Texas. Stock solutions of these materials were made
250 1500
x Further investigations are needed to better understand the
Figure 7: Nuclei of A549 cells treated with SWNT mechanisms by which nanoparticles induce apoptosis and

Mean intensity of mitochondrial stain

as a suspension in 1% Acacia Gum (AcGum, Sigma Cat # G 200 1200
also to decipher other cellular pathways that are affected

Mean intensity of permeability stain

about 3 hours post-exposure of cells to nanoparticles, 9752). show apoptosis morphology by nanoparticles.
(mean ± SD)
depending on the nature of cellular targets selected for The cells growing in 96 well plates were treated with varying 150 ** 900

(mean ± SD)
x

studying. Also, all of the steps involved in sample preparation doses of the nanoparticles for 24 hrs. The cells were in a 5% 100

for HCS can be automated, providing for a very efficient way CO2 incubator at 37O C during this incubation.
600
**

to screen for a variety of targets or nanoparticles in a short Staining with Hoechst-33342 (0.2 Pg/mL or 1 Pg/mL),
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significant effect on cell membrane permeability. The cell cycle
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staining by Hoechst-33342 towards a sub G0/G1 cell cycle
status in a dose dependent manner. This observation suggests
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Tre atm ent (P g/m L)
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Effect of varying doses of different nanoparticles on the multiwall carbon nanotubes and nano-onions on human
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E (red) are beads with no SWNT and panels B (blue), D (green)
treated and stained as described earlier. Data is mean ± SD from processed as described earlier. Data is mean ± SD from 16 wells
& F (red) are beads mixed with 1780 Pg/mL SWNT. Panel G
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shows the fluorescent intensity of the beads with varying
SD from 16 wells. ** indicates data that was statistically MWNT had an effect on mitochondrial potential staining, while
concentrations of SWNT in the red wavelength region. Similar
significant (p < 0.05 by Student’s t test) from AcGum and media C60 Fullerene had no effect at this high dose also.
plots were obtained for the other wavelength regions also.
controls.