interference of engineered nanoparticles with in vitro toxicity assays
TRANSCRIPT
INORGANIC COMPOUNDS
Interference of engineered nanoparticles with in vitrotoxicity assays
Alexandra Kroll • Mike Hendrik Pillukat •
Daniela Hahn • Jurgen Schnekenburger
Received: 14 October 2011 / Accepted: 1 March 2012 / Published online: 11 March 2012
� Springer-Verlag 2012
Abstract Accurate in vitro assessment of nanoparticle
cytotoxicity requires a careful selection of the test systems.
Due to high adsorption capacity and optical activity,
engineered nanoparticles are highly potential in influencing
classical cytotoxicity assays. Here, four common in vitro
assays for oxidative stress, cell viability, cell death and
inflammatory cytokine production (DCF, MTT, LDH and
IL-8 ELISA) were assessed for validity using 24 well-
characterized engineered nanoparticles. For all nanoparti-
cles, the possible interference with the optical detection
methods, the ability to convert the substrates, the influence
on enzymatic activity and the potential to bind proinflam-
matory cytokines were analyzed in detail. Results varied
considerably depending on the assay system used. All
nanoparticles tested were found to interfere with the optical
measurement at concentrations of 50 lg cm-2 and above
when DCF, MTT and LDH assays were performed. Except
for Carbon Black, particle interference could be prevented
by altering assay protocols and lowering particle concen-
trations to 10 lg cm-2. Carbon Black was also found to
oxidize H2DCF-DA in a cell-free system, whereas only
ZnO nanoparticles significantly decreased LDH activity. A
dramatic loss of immunoreactive IL-8 was observed for
only one of the three TiO2 particle types tested. Our results
demonstrate that engineered nanoparticles interfere with
classic cytotoxicity assays in a highly concentration-, par-
ticle- and assay-specific manner. These findings strongly
suggest that each in vitro test system has to be evaluated for
each single nanoparticle type to accurately assess the
nanoparticle toxicity.
Keywords Engineered nanoparticles � Cytotoxicity
assays � Interference � Cytokine adsorption
Abbreviations
H2DCF-DA 20,70-Dichlorodihydrofluorescein diacetate
DCF 20,70-Dichlorofluorescein
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazoliumbromide)
INT (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-
phenyl tetrazolium chloride)
MTS 3-(4,5-Dimethylthiazole-2-yl)-5-
(3-carboxymethoxyphenyl)-2-
(4-sulfophenyl)-2H-tetrazoliumin
Introduction
The increased global production of engineered nanoparti-
cles (NPs) and their application in various fields require a
detailed understanding of their potential toxicity. In vitro
and in vivo studies demonstrated that NPs may display a
higher toxic potential than fine-sized particles of identical
This article is published as a part of the Special Issue
‘‘Nanotoxicology II’’ on the ECETOC Satellite workshop,
Dresden 2010 (Innovation through Nanotechnology and
Nanomaterials ? Current Aspects of Safety Assessment and
Regulation).
Alexandra Kroll and Mike Hendrik Pillukat contributed equally to
this article.
A. Kroll � M. H. Pillukat � D. Hahn � J. Schnekenburger (&)
Biomedical Technology Center, Westfalische
Wilhelms-Universitat, Albert-Schweitzer-Campus 1 A14,
48149 Munster, Germany
e-mail: [email protected]
Present Address:A. Kroll
Eawag, Swiss Federal Institute of Aquatic Science
and Technology, Dubendorf, Switzerland
123
Arch Toxicol (2012) 86:1123–1136
DOI 10.1007/s00204-012-0837-z
composition (Duffin et al. 2007; Landsiedel et al. 2010;
Oberdorster et al. 2005; Singh and Nalwa 2007). Apart
from size and chemical composition, a variety of other
physicochemical properties including crystallinity (Sayes
et al. 2006; Wang et al. 2008), agglomeration state
(Grassian et al. 2007), surface coating (Cho et al. 2007; Pan
et al. 2009), shape (Rothen-Rutishauser et al. 2010) and
microporosity (Rabolli et al. 2010) have recently been
identified as critical determinants of NP toxicity. More-
over, evidence is accumulating that NP toxicity is driven
by interplay of several of these properties and is difficult to
predict a priori (Johnston et al. 2009; Rabolli et al. 2010;
Shaw et al. 2008, Kroll et al. 2011).
Evaluating NP toxicity therefore requires a testing
strategy that includes a detailed physicochemical charac-
terization of each individual particle type as well as a set of
in vitro assays that allow for a rapid screening of the vast
number of existing and newly developed NP variants.
Currently, however, standardized in vitro techniques to
accurately assess NP toxicity have not yet been established.
Recent studies suggest that classical cell-based assays
designed for testing chemicals may not provide reliable
data since nanomaterials can interfere with assay compo-
nents and readout systems (Kocbach et al. 2008; Kroll et al.
2009; Monteiro-Riviere et al. 2009).
Due to their large surface area and high surface energy,
nanoparticles can adsorb assay reagents or reporter
dyes, thereby distorting the assay outcome. For carbon
nanomaterials, such as single-walled carbon nanotubes
(SWCNTs) or Carbon Black (CB), adsorption of MTT-
formazan, a reporter dye that indicates metabolic activity in
MTT assays, has been described by different groups of
investigators (Casey et al. 2007; Monteiro-Riviere and
Inman 2006; Monteiro-Riviere et al. 2009; Worle-Knirsch
et al. 2006). Additionally, SWCNTs seem to adsorb
essential nutrients from cell culture media resulting in
indirect cytotoxic effects (Casey et al. 2008; Guo et al.
2008). In both cases, the high adsorption capacity of carbon
nanomaterials generates false-positive data resulting in an
overestimation of their toxicity; notably, CB also seems to
adsorb proinflammatory cytokines leading to false-negative
results in some immunoassays and, consequently, to an
underestimation of its potential toxicity (Brown et al. 2010;
Kocbach et al. 2008). This demonstrates NP interference
with assay systems as cause for contradictory testing results.
Verifying cytotoxicity data with at least two or more
independent test systems as it has been suggested earlier
(Monteiro-Riviere et al. 2009) may thus not be sufficient to
eliminate misleading results and incorrect interpretations.
Recent studies indicate that also metal oxide NPs
interact with assay components or reporter dyes of in vitro
test systems. Ultrafine superparamagnetic iron oxide NPs
seem to interfere with MTS-based cell viability assays
(Doak et al. 2009), a confounding factor that results in
an underestimation of their potential toxicity. Moreover,
superparamagnetic iron oxide NPs can cause significant
changes in the cell culture medium such as depletion of
Cl- ions or denaturation of proteins that, in turn, can result
in toxic effects on cells (Mahmoudi et al. 2010). Finally,
Veranth et al. have found TiO2 and SiO2 to cause artifacts
in immunoassays by adsorbing the proinflammatory cyto-
kine IL-6 (Veranth et al. 2007).
Additional challenges for NP cytotoxicity screening
arise from the intrinsic optical absorbance of NP disper-
sions. Because most of the classic in vitro toxicity assays
are based on optical readouts, the high absorption or
scattering of nanoparticles may interfere with the detection
method. Carbon nanomaterials are known to absorb light of
the visible spectrum (Wells and Smith 1941) and may
therefore generate an absorbance at the same wavelength as
that used to detect the colored assay product. Metal oxide
NPs frequently found in sunscreens, such as TiO2 and ZnO,
absorb and scatter UV radiation as well as visible light
(Wolf et al. 2001) and may possibly influence absorbance
readouts of colorimetric assays. In fact, TiO2 NPs have
been recently shown to change the color and increase the
opalescence of experimental resin matrices (Yu et al.
2009). However, the influence of NPs on optical detection
is difficult to estimate in advance, as optical activity does
not only depend on the chemical composition but also on
other properties such as particle size, shape and crystal-
linity as well as on the dispersion agent. A number of NPs
also seem to interact directly with fluorescent dyes, thereby
altering their emission intensity. Silver NPs, for instance,
have been shown to quench fluorescent dyes presumably by
binding them onto their surface (Sabatini et al. 2007). Gold
nanoparticles have been found to quench DCF fluorescence
upon NP-induced oxidative stress (Pfaller et al. 2010).
Dextran-coated iron oxide NPs displayed interference with
the fluorescence emission of DCFH. The interference was
more pronounced with Fe3O4 than with Fe2O3 NPs, dem-
onstrating that even differences in the oxidation state can
impact the test results (Doak et al. 2009). Testing NPs with
established assays is therefore challenging and requires a
careful evaluation of the test systems applied.
In this study, we utilized a set of 24 well-characterized
engineered nanoparticles and assessed their possible
interference with classical cytotoxicity assays. We selected
frequently used in vitro assays that determine four different
cytotoxic endpoints (oxidative stress (DCF), metabolic
activity (MTT), cell death (LDH) and immune responses
(IL-8 ELISA)) and evaluated their suitability for measuring
toxic effects of NPs on cells. We developed standardized
protocols for the experimental setup including the prepa-
ration of NP dispersions, exposure conditions and test
procedures. For each of the 24 NPs, we tested its possible
1124 Arch Toxicol (2012) 86:1123–1136
123
interference with the optical detection methods, its ability
to convert the substrate, its influence on the lactate dehy-
drogenase activity as well as its adsorption of cytokines.
The NPs represented inorganic compounds manufactured
for a wide range of industrial products including three
different TiO2 particle types and, additionally, CB. Within
the framework of the German project NanoCare, the NPs
had been previously characterized in detail regarding their
physicochemical properties (Landsiedel et al. 2010; Nano-
Care Project 2009).
Materials and methods
Nanoparticles
The engineered NPs were part of the BMBF project
NanoCare materials and were thoroughly characterized
regarding relevant physicochemical properties such as size
distribution, surface area, crystallinity, crystal phase and
solubility (Landsiedel et al. 2010; NanoCare Project 2009).
Crystalline quartz particles (DQ12, mean particle size of
5 lm) were kindly provided by Evonik Degussa GmbH
(Marl, Germany), and fine-sized ZnO particles (mean
particle size of 350 nm) were purchased from Sigma-
Aldrich Chemie GmbH (Munich, Germany).
Chemicals
If not otherwise stated, all chemicals were obtained from
Sigma-Aldrich Chemie GmbH (Munich, Germany). 20,70-Dichlorodihydrofluorescein diacetate (H2DCF-DA) was
obtained from Invitrogen GmbH (Karlsruhe, Germany).
Sodium bicarbonate and sodium pyruvate were from Lonza
AG (Koln, Germany). Dulbecco’s modified Eagle’s med-
ium (DMEM, E15-877), RPMI 1640 (E15-848) and FBS
Gold (A15-151) were supplied by PAA Laboratories
GmbH (Pasching, Germany). Phenol red–free MEM was
obtained from Biochrome AG (Berlin, Germany).
Cell lines and culture conditions
A549 cells were obtained from the American Type Culture
Collection (ATCC, Rockville, USA). Their authenticity
was determined by DSMZ (German Collection of Micro-
organisms and Cell Cultures) via DNA fingerprinting.
Cultured cells were propagated at 37 �C and 5 % CO2.
Cells were grown to confluence within three to 4 days in
DMEM supplemented with 10 % v/v heat-inactivated fetal
calf serum (FBS Gold) and 2 mM L-glutamine and then
transferred to new culture plates at a dilution of 1:6. Cells
were cultured for a maximum of 20 passages with regular
testing for mycoplasma contamination by PCR. To assure
constant cell quality, the cell cycle state and metabolic
activity of the cell cultures were monitored prior to each
experiment with propidium iodide staining followed by
flow cytometry or with the MTT assay. To prepare cell
monolayers for testing NP interference, cells were seeded
in 96-well plates at a density of 3 9 104 cells per well in a
total volume of 100 ll and incubated at 37 �C, 5 % CO2
for 24 h. To avoid any influence of the possible photocat-
alytic activity of the NPs, DCF, MTT and LDH assays
were performed in the absence of UV light.
For immunoassays, cells were seeded at a density of
2 9 104 cells per well in a total volume of 100 ll and
cultured as described above. After centrifugation (15 min,
5009g, RT), supernatants were removed from the assay
plates, diluted 20-fold in DMEM/10 % FBS Gold and
stored at -20 �C until assayed by immunoassays.
Sterilization and dispersion of nanoparticles in cell
culture medium
NPs were sterilized and dispersed according to a stan-
dardized protocol as described previously (Schulze et al.
2008). In particular, NP aliquots of 19.2 mg in powder
form or predispersed as supplied by the manufacturer were
transferred into sterile snap-lid glasses together with a
small magnetic stir bar and subsequently exposed to 30-Gy
gamma irradiation. The dispersions were stirred at 900 rpm
for 1 h at room temperature in 6 ml of DMEM/10 % FBS.
Dilutions of this stock dispersion were prepared immedi-
ately and stirred for 24 h at 900 rpm at room temperature.
All NP dispersions (10 lg cm-2 in DMEM/10 % FBS)
were tested to be free of endotoxins using the Limulus
Amebocyte Lysate (LAL) Kinetic-QCL� kit (50-650U),
Lonza GmbH, Wuppertal, Germany.
Interference with the detection of oxidative stress
The formation of reactive oxygen species (ROS) as indi-
cators of oxidative stress is commonly detected using
the fluorescein derivative H2DCF-DA (H2DCF-DA: 20,70-dichlorodihydrofluorescein diacetate; Jakubowski and
Bartosz 2000). H2DCF-DA penetrates cell membranes, is
hydrolyzed by cellular esterases and is converted via
reactive oxygen species (ROS) to the fluorescent oxidation
product DCF. The interference of NPs with the optical
detection of DCF fluorescence was assessed by replacing
the assay substrate H2DCF-DA by defined amounts of
fluorescent DCF. Cell monolayers prepared as described
above were incubated for 1 h at 37 �C with 100 ll NP
dispersions at concentrations of 0.01, 0.1, 1, 10, 50 or
100 lg cm-2. As control, stirred cell culture medium
(100 ll per well) was used to replace the NP dispersions.
Different concentrations of DCF (100, 50, 10, 1, 0.1 or 0.01
Arch Toxicol (2012) 86:1123–1136 1125
123
nM) diluted in Krebs–Ringer buffer (KRB; pH 7.4,
114.2 mM NaCl, 3 mM KCl, 1.5 mM K2HPO4 9 3 H2O,
10 mM HEPES, 3.996 mM D-glucose, 1.405 mM CaCl2,
2.562 mM MgCl2) were added either directly or after the
cells had been washed with KRB. Fluorescence was
immediately monitored in a spectrophotometer (excitation
485 nm, emission 520 nm; NOVOstar and FLUOstar,
BMG Labtech GmbH, Offenburg, Germany). Each exper-
iment was repeated at least seven times with seven
replications.
To test the catalytic activity of NP dispersions in terms
of H2DCF-DA oxidation, empty 96-well plates were
incubated with NP dispersions (10, 5, 1, 0.1 or 0.01 lg
cm-2) or stirred cell culture medium (control) and incu-
bated for 1 h at 37 �C. After washing with 100 ll KRB, the
H2DCF-DA assay solution was added and incubated for
1 h at 37 �C. Subsequently, the plates were washed twice
with KRB, and fluorescence was recorded twice, immedi-
ately and after an additional incubation for 3 h at 37 �C.
Each experiment was repeated at least three times with
seven replications.
Interference with the detection of metabolic activity
The MTT assay determines cellular metabolic activity by
the reduction of the yellow tetrazolium salt MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) to
a purple MTT-formazan. The interference of NP disper-
sions with the optical detection of MTT-formazan was
assessed using different mixtures of the reaction product
MTT-formazan (1.2, 0.8, 0.24, 0.024 and 0.0024 mM)
with the substrate (2.4 mM oxidized MTT) to cover the
whole spectrum of light absorption. MTT-formazan was
obtained by incubating A549 cell monolayers with oxi-
dized MTT. In particular, cell monolayers grown as
described above were washed twice with 100 ll PBS and
incubated with 2.4 mM oxidized MTT in 110 ll PBS for
2 h. To dissolve the MTT-formazan crystals, 100 ll of
15 % SDS/50 % DMSO was added; 96-well plates were
shaken at 300 rpm overnight at 37 �C, and the superna-
tants were pooled for further experiments. To verify that
MTT had been fully reduced, cell supernatants were
recovered after incubation with oxidized MTT and added
to fresh cell monolayers. Different NP concentrations of
0.01, 0.1, 1, 10, 50 and 100 lg cm-2 in dispersion were
added to cell monolayers (see above) and incubated for
24 h at 37 �C. Stirred cell culture medium (100 ll per
well) was used as control. Mixtures of reduced and oxi-
dized MTT (100 ll per well) were directly added either
after NP incubation or after the cells were washed with
KRB. Light absorption was measured in a spectropho-
tometer (excitation 485 nm, emission 520 nm; NOVOstar
and FLUOstar, BMG Labtech GmbH; Offenburg,
Germany) at 550 nm and for reference at 670 nm. Each
experiment was repeated three times with seven
replications.
The influence of NP dispersions on the reduction of
MTT was determined in empty wells incubated with NP
dispersions (10, 5, 1, 0.1 and 0.01 lg cm-2) or stirred cell
culture medium (control) and washed with KRB The
standard MTT solution (2.4 mM MTT) was added and
incubated for 3 h at 37 �C. To dissolve the MTT-formazan
crystals, 100 ll of 15 % SDS/50 % DMSO was added and
shaken overnight at 37 �C. Light absorption was measured
at 550 nm. Each experiment was repeated three times with
seven replications.
Interference with the measurement of cell death
Cell death is usually quantified by the measurement of
lactate dehydrogenase (LDH) activity in cell supernatants.
LDH reduces INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-
phenyl tetrazolium chloride) in the presence of NADH ?
H? (reduced b-nicotinamide adenine dinucleotide) yielding
a red water-soluble formazan, which is quantified by the
measurement of light absorption (Nachlas et al. 1960). The
interference of NP dispersions with the optical detection of
INT reduction was analyzed with different mixtures of
reduced and oxidized INT. Reduced INT was obtained by
incubating supernatants of A549 cell monolayers prepared
as described above with oxidized INT according to Kor-
zeniewski and Callewaert (Korzeniewski and Callewaert
1983). To verify that INT had been entirely reduced, part of
the reaction product was incubated according to the same
protocol with fresh cell supernatant and reaction mixture
but without oxidized INT. Cell monolayers prepared as
described above were then exposed to NP dispersions at
concentrations of 0.01, 0.1, 1, 10, 50 and 100 lg cm-2 for
24 h at 37 �C. Stirred cell culture medium (100 ll per
well) was used as control. Supernatants (50 ll) were
transferred to empty 96-well plates and mixtures of
reduced INT (1.75, 1.16, 0.35, 0.035 and 0.0035 mM,
respectively) with 3.5 mM oxidized INT were added. The
light absorption at 492 nm was monitored immediately.
Each experiment was repeated three times with seven
replications.
The catalytic activity of NPs was tested in cell-free
wells pretreated with NP dispersions (10, 5, 1, 0.1 and
0.01 lg cm-2, respectively) or with stirred cell culture
medium (control). Supernatants (50 ll) were mixed with
50 ll of the LDH assay solution (3.5 g l-1 INT,
0.089 g l-1 PMS and 0.9 g l-1 NAD in 0.2 M Tris-Base
pH 8.2 and 5 g l-1 lactic acid). Light absorption was
measured continuously for 1 h at 37 �C in a spectropho-
tometer. Each experiment was repeated three times with
seven replications.
1126 Arch Toxicol (2012) 86:1123–1136
123
Bovine LDH (Sigma-Aldrich Chemie GmbH, Munich,
Germany) was used to determine the influence of NP dis-
persions on LDH activity. NP dispersions or stirred cell
culture medium (control) were mixed with 0.3 U ml-1 LDH
in cell-free wells (100 ll per well) and incubated for 24 h at
37 �C. Supernatants (50 ll) were transferred to new 96-well
plates, and 50 ll of the LDH assay solution was added. Light
absorption at 492 nm was recorded continuously for 1 h at
37 �C in a spectrophotometer. Each experiment was repe-
ated three times with seven replications.
Determination of IL-8 with the enzyme-linked
immunosorbent assay
A549 cells were seeded on 96-well plates and incubated
with NP dispersions (1, 10 and 50 lg cm-2, respectively),
dispersed quartz DQ12 as positive and stirred cell culture
medium as negative control. Cytokine levels in the diluted
samples were determined using the Human IL–8 Opt-
EIATM–ELISA-Kit (BD Biosciences; Heidelberg, Germany)
according to the manufacturer’s protocol with minor
modifications: Dilution of samples (1:20 for the determi-
nation of IL-8 in supernatants from cell cultures, undiluted
for samples from cell-free assays) and of protein standard
was carried out in DMEM/10 % FBS Gold (PAA Labo-
ratories GmbH; Pasching, Germany) and not in dilution
buffer (PBS/10 % FBS Gold). The IL-8 protein standards
were diluted from 300 pg ml-1 down to 4.69 pg ml-1. The
ELISA plates were washed by an automated Opsys Plate
Washer MW (Dynex Technologies Inc., Chantilly, VA,
USA). Absorbance was measured at 450 nm with the plate
reader FLUOstar OPTIMA (BMG Labtech GmbH; Off-
enburg, Germany). Each experiment was repeated twice
with three replications.
Interference of NPs with immunoassays
Cell-free experiments were carried out with different con-
centrations of recombinant IL-8 protein standard (ranging
from 4.69 to 300 pg ml-1, from the Human IL-8 Opt-
EIATM–ELISA-Kit) instead of cells and without medium
exchange before the addition of nanoparticles (50 lg
cm-2) or stirred control medium, respectively. After
incubation and centrifugation (15 min, 5009g, RT), the
supernatants were removed from the assay plates, and IL-8
concentration was determined by ELISA. Two independent
experiments were performed with each of the 24 nano-
particles and controls.
Selected NPs (TiO2 1–3 NPs, CB) were tested for
cytokine adsorption at three concentrations (1, 10 and
50 lg cm-2) and with four different concentrations of
recombinant IL-8 (18.25–150 pg ml-1). These experi-
ments were performed twice with two replications each.
Calculations and statistical analyses
Results are presented as mean values with standard devi-
ations. Statistical significance was compared to the
respective on-plate negative control using Student’s t test
or ANOVA according to Tukey for p = 0.05.
Results
For the analysis of NP interference with common in vitro
toxicity test systems, we assessed 24 engineered NPs using
cytotoxicity assays monitoring oxidative stress (DCF), cell
death (LDH), cellular metabolic activity (MTT) and the
release of the proinflammatory cytokine IL-8 (ELISA). All
experiments were performed with A549 cells that are
commonly used as an in vitro model for lung toxicity.
These cells display features of type II lung alveolar epi-
thelial cells (Stearns et al. 2001). The NPs used in this
study included particles with the same chemical composi-
tion but with different physical properties, for example,
three types of TiO2 or ZrO2 NPs (NanoCare Project 2009).
Fine-sized ZnO (FZO) and crystalline quartz (DQ12) par-
ticles were used as reference materials. Dispersions of
nanoparticles were prepared in a standardized procedure as
previously described (Schulze et al. 2008). Moreover, each
NP dispersion was tested to be free of lipopolysaccharides,
that is, endotoxins that induce inflammatory responses or
oxidative stress in cultured cells (Esch et al. 2010; Schulze
et al. 2008).
To obtain a more comprehensive view of the possible
influences of NPs on the test systems, we used the complete
set of 24 engineered NPs and tested each NP for optical
interference, for interaction with the substrates, for the
inhibition of enzymes (LDH) and for adsorption of cyto-
kines (IL-8) from cell supernatants.
Optical interference
The fluorescence-based DCF assay determines the forma-
tion of intracellular reactive oxygen species (ROS) via
oxidation of the non-fluorescent substrate H2DCF-DA to
the fluorescent DCF product. To investigate the interfer-
ence of NP dispersions with the fluorescence measurement
of DCF, we used dilutions of fluorescent DCF covering the
full detection range of the spectrofluorometer and deter-
mined their fluorescence emission at 520 nm in medium
or in the presence of NP dispersions (0.1, 1, 10 and
50 lg cm-2) by following the assay protocols (for details,
see Materials and methods). All NP types tested interfered
with the detection of DCF fluorescence at concentrations of
10 lg cm-2 (32 lg ml-1) and above with CB displaying
the strongest influence observed. Interference of CB and
Arch Toxicol (2012) 86:1123–1136 1127
123
TiO2 3 NPs with the measurement of DCF fluorescence is
shown exemplarily in Fig. 1a, b. DCF fluorescence was
significantly reduced in the presence of 1–50 lg cm-2 NP
dispersions of CB (Fig. 1a) and Al–Ti–Zr 3 (data not
shown), irrespective of the DCF concentrations applied. NP
dispersions of TiO2 3 and all other NPs tested displayed
interference at particle concentrations of 10–50 lg cm-2
(see Fig. 1b as an example).
To minimize particle interference, the classical DCF
assay protocol was modified and NP dispersions were
removed by washing the cell monolayers prior to the
incubation with DCF (for details, see Materials and
methods). These assay adaptations nearly eliminated DCF
signal alteration when particle concentrations of
1 lg cm-2 (CB, Fig. 1c) or 10 lg cm-2 and below were
used (TiO2 3, Fig. 1d and all other NPs tested, data not
shown).
The MTT assay is a dye-based assay that determines
cellular metabolic activity by colorimetric quantification.
In metabolically active cells, the yellow tetrazolium
substrate MTT is reduced to a purple insoluble MTT-for-
mazan and results are obtained by quantitation of colori-
metric absorption. We assessed the interference of the 24
engineered NPs with the measurement of MTT and MTT-
formazan. To this end, mixtures of oxidized MTT and
MTT-formazan that covered the whole spectrum of light
absorption from the yellow reactant to the purple product
were prepared and measured in the presence or absence of
NP dispersions with different particle concentrations. In
Fig. 2a, b, MTT light absorption in the presence of CB and
TiO2 3 is shown exemplarily. Light absorption of each
MTT/MTT-formazan mixture was strongly distorted in the
presence of all nanoparticle dispersions tested when parti-
cle concentrations of 10 lg cm-2 and above were used
(Fig. 2a, b). The effect was most pronounced in mixtures
containing higher concentrations of MTT-formazan (0.8
and 1.2 mM). We therefore modified the MTT assay pro-
tocols and applied the NP dispersions for 24 h but washed
them from the cell monolayer prior to the incubation with
MTT. CB still interfered with light absorption of MTT at
Fig. 1 Interference of CB (a, c) and TiO2 3 (b, d) NPs with DCF
fluorescence. Fluorescence of five different DCF concentrations was
measured in the presence of A549 cells pre-incubated for 1 h with
dispersions of four different NP concentrations in DMEM/10 % FBS.
Serum-containing medium (DMEM/10 % FBS) was used as control.
In a and b, the experimental procedure followed the classic DCF
protocol; in c and d, an additional washing step was included before
the addition of DCF. Mean values with standard deviations were
obtained from seven independent experiments with seven replications
each
1128 Arch Toxicol (2012) 86:1123–1136
123
10 lg cm-2 and above (Fig. 2c), but for all other NPs
tested, these assay adaptations eliminated interference with
the measurement of MTT/MTT-formazan mixtures up to
particle concentrations of 10 lg cm-2 (see Fig. 2d as an
example).
In LDH assays, the yellow tetrazolium salt INT is used to
detect active LDH released into the cell culture medium from
damaged cells. LDH converts lactate to pyruvate, thereby
generating NADH ? H? that, in turn, is used to reduce INT
to a red formazan. Interference of NPs with INT light
absorption was determined with mixtures of reduced and
oxidized INT (for details, see Materials and methods). For all
NPs, interference could be detected with particle concen-
trations of 50 lg cm-2 but not with concentrations of
10 lg cm-2 and below (see representative Fig. 3).
Influence on substrate conversion
We determined the ability of the NP dispersions to convert
the substrates H2DCF-DA, MTTox and INTox. To this
end, the substrates were added to cell-free 96-well plates
pre-incubated with NP dispersions at different particle
concentrations. When H2DCF-DA was incubated for 1 h,
we could not detect DCF fluorescence signals with any of
the particles used (see Fig. 4a, b displaying representative
examples). However, 4 h after the addition of H2DCF-DA,
we detected DCF signals in the presence of CB dispersions
that increased in a concentration-dependent manner
(Fig. 4a). All other NPs had no effect (Fig. 4b).
Influence on enzymatic activity
Because of their large surface area and high surface energy,
NPs may be able to adsorb enzymes on their surface and
possibly inactivate the adsorbed protein.
We therefore analyzed the complete set of NPs regard-
ing their effects on the enzymatic activity of lactate
dehydrogenase. LDH assays were performed in cell-free
wells containing bovine LDH in the presence of NP dis-
persions with different particle concentrations (0.01, 0.1, 1,
5 and 10 lg cm-2). At particle concentrations of up to
5 lg cm-2, we could not detect any significant influence
Fig. 2 Interference of CB (a, c) and TiO2 3 (b, d) NPs with MTT-
formazan light absorption. NP dispersions with four different particle
concentrations in DMEM/10 % FBS were added to A549 monolayers
and incubated for 24 h. Serum-containing medium (DMEM/10 %
FBS) was used as control. Mixtures of reduced and oxidized MTT
were added either directly (a, b) or after washing with KRB (c, d).
Results are displayed as arbitrary units [AU]. Mean values and
standard deviations were obtained from three independent experi-
ments with seven replications each
Arch Toxicol (2012) 86:1123–1136 1129
123
on LDH activity. However, at 10 lg cm-2, a slight but
significant inhibition of LDH activity was observed with
ZnO NPs (Fig. 5, NZO), whereas no other NPs tested
displayed any significant effect on LDH activity. Inhibition
of LDH activity could also be observed in the presence of
fine-sized ZnO particles, suggesting a composition-depen-
dent rather than a size-/surface-dependent effect (Fig. 5,
FZO).
Effects of NPs on cellular IL-8 levels
The levels of the proinflammatory peptide IL-8 were
assessed in A549 cells after a 24-h exposure to 50 lg cm-2
of each of the 24 engineered NPs and reference particles
(FZO, DQ12). For nine out of 24 NPs, a significant
induction of cellular cytokine production was observed
(Fig. 6a–d). Strikingly, after exposure to TiO2 2 NPs, the
cells displayed IL-8 secretion below control levels,
whereas in cells exposed to TiO2 1, IL-8 production
resembled control levels (Fig. 6b). TiO2 1 and TiO2 2 NPs
are identical particles from the same production process
and differ only by predispersion. TiO2 1 was obtained in
powder form, whereas TiO2 2 was predispersed by the
supplier. TiO2 3 was of similar size but of different crys-
talline structure and particle morphology. In cells stimu-
lated with TiO2 3, IL-8 levels were even higher than those
observed for the reference DQ12 and higher than any other
NPs tested (Fig. 6b).
Fig. 3 Interference of CB (a) and TiO2 3 (b) NPs with INT-formazan
light absorption. Light absorption of INTred/INTox mixtures was
measured in the presence of A549 cells pre-incubated with NP
dispersions of four different particle concentrations in DMEM/10 %
FBS. Results are displayed as arbitrary units [AU]. Mean values and
standard deviations were obtained from three independent experi-
ments with seven replications each. *Significantly different from
control (DMEM/10 % FBS) for p = 0.05
Fig. 4 NP-mediated conversion of H2DCF-DA in a cell-free assay.
Cell-free microtiter plates were pre-incubated with CB (a) or TiO2 3
(b) dispersions of five different particle concentrations, washed and
incubated with H2DCF-DA for 1 and 4 h, respectively. Serum-
containing medium (DMEM/10 % FBS) was used as control. Mean
values and standard deviations were obtained from three experiments
with seven replications each
1130 Arch Toxicol (2012) 86:1123–1136
123
Fig. 5 Inhibition of LDH
activity in the presence of NPs.
Particle dispersions (10 lg
cm-2) were mixed with bovine
LDH and incubated for 24 h.
LDH activity was determined
with INT followed by the
measurement of INT-formazan
absorption. Mean values and
standard deviations displayed as
% of control (DMEM/10 %
FBS) were obtained from three
independent experiments with
seven replications each.
*Significantly different from
control for p = 0.05
Fig. 6 IL-8 levels in A549 culture supernatants after 24-h exposition to nanoparticles. All particles were tested in triplicate in two independent
assay series each. Statistical significance was determined by Student’s t test with p \ 0.05 considered as significant (*)
Arch Toxicol (2012) 86:1123–1136 1131
123
Effects of NPs on IL-8 levels in cell-free systems
To test the influence of NPs on measurable IL-8 protein
levels, we determined the absorption of IL-8 ELISA stan-
dards with seven different IL-8 concentrations (from 4.69
to 300 pg ml-1) in the presence of NP dispersions with
particle concentrations of 50 lg cm-2 in a cell-free system.
Of the 24 NPs tested, 23 NPs did not display any sig-
nificant effect on the IL-8 standard curve after incubation
for 24 h (Fig. 7a–d). In the presence of TiO2 2, however, a
dramatic loss of immunoreactive IL-8 was observed with
all IL-8 standard concentrations tested (Fig. 7b). In con-
trast, TiO2 1 from the same production process (but not
predispersed) did not influence measurable IL-8 protein
levels. These results resemble those observed for the
detection of cellular IL-8 levels (Fig. 6b) and suggest an
adsorption of IL-8 by TiO2 2 NPs.
Adsorption of IL-8 by NPs
We further investigated the NP concentration-dependent
potential interleukin binding to a selection of NP types.
Dispersions of TiO2 NPs (1–3) and CB with different
particle concentrations (1, 10 and 50 lg cm-2) were added
to four different IL-8 protein concentrations in medium and
incubated for 24 h prior to the measurement by ELISA. As
shown in Fig. 8, we did not observe the depletion of
immunoreactive IL-8 with any of the CB, TiO2 1 and 3
dispersions tested (Fig. 8a–c).
As expected, measurable IL-8 levels were dramatically
reduced in dispersions containing 50 lg cm-2 TiO2 2 NPs
(Fig. 8d), presumably due to the adsorption of the cytokine
on the particle surface. Only a weak decrease in detectable
IL-8 protein was observed for dispersions with 10 lg cm-2
TiO2 2, and particle concentrations of 1 lg cm-2 did not
affect IL-8 measurements in a significant manner (Fig. 8d).
Discussion
In this study, we tested four classical in vitro assays for
their validity to assess NP toxicity and optimized the pro-
tocols to avoid particle interference. We used 24 well-
characterized engineered NPs (Kroll et al. 2011) and
investigated each NP for its interference with the optical
detection methods as well as its interaction with the dyes,
Fig. 7 Screening of nanoparticles for ELISA interference. Different
particle dispersions (50 lg-2) were incubated for 24 h together with
recombinant human IL-8 protein standards. Graphs shown represent
typical resulting standard curves of one out of two independent assay
series with seven different concentrations ranging from 300 to
4.69 pg ml-1 IL-8 with or without nanoparticles in DMEM ? 10 %
fetal bovine serum plus appropriate controls
1132 Arch Toxicol (2012) 86:1123–1136
123
enzymes or peptides used in DCF, MTT, LDH and IL-8
ELISA assays. Although a number of studies have shown
interaction of carbon-based nanomaterials with reporter
dyes used in cell viability assays (Belyanskaya et al. 2007;
Casey et al. 2007; Monteiro-Riviere et al. 2009; Worle-
Knirsch et al. 2006), only very limited information is
available for inorganic compounds and for their possible
interference with optical detection. Recently, Pfaller et al.
reported enhanced fluorescence intensities when cell-free
DCF assays were performed in the presence of 4.5-nm gold
NPs. This spectral interference was thought to result from
an interaction of the excited fluorophores with free elec-
trons in the metals (Pfaller et al. 2010). Here, we observed
an apparent reduction of DCF fluorescence transmission
with all of the 24 nanoparticles tested, already at particle
concentrations from 10 lg cm-2. The effect was most
pronounced in the presence of CB and can easily be
explained by its ability to absorb light in the visible spec-
trum (Wells and Smith 1941). Since both the excitation
(485-nm) and the emission (520-nm) wavelengths of the
DCF marker lie within the spectrum of visible light, CB
may absorb not only the emitted DCF fluorescence but also
the excitation energy, thereby preventing excitation of the
fluorophore. The other nanoparticles represented metal
oxides, metal hydroxides and metal carbonates that give
whitish or yellowish opaque dispersions in serum-con-
taining cell culture medium. The decreased DCF fluores-
cence signal measured in the presence of these NP
dispersions is therefore likely to result to a larger extent
from light scattering rather than from absorption. Remov-
ing NPs from the solution by including washing or cen-
trifugation steps before measuring DCF fluorescence is
therefore essential to avoid methodological artifacts. Given
the fact that the interference of NPs is concentration
dependent and that NPs cannot be removed entirely from
the solution, NP dispersions applied should also be limited
to concentrations that do not interfere with the measure-
ment of DCF signals. For the set of NPs tested here, a
maximum concentration of 10 lg cm-2or 32 lg ml-1 (for
CB 1 lg cm-2) can be used without optical interference
when performing an optimized DCF assay protocol. Since
interference with the fluorimetric measurement depends on
Fig. 8 Testing of different concentrations of selected nanoparticles
for ELISA interference. Different nanoparticle dispersions in three
concentrations (1, 10 and 50 lg cm-2) were incubated for 24 h with
recombinant human IL-8 protein standard concentrations. Data shown
represent four assays (a–d) with four different concentrations ranging
from 150 to 18.25 pg ml-1 IL-8 with or without nanoparticles
dispersions (performed four times). Statistical significance was
determined by Student’s t test with p \ 0.05 considered as significant
(*) and with p \ 0.001 considered as highly significant (**)
Arch Toxicol (2012) 86:1123–1136 1133
123
the physico-chemical properties of the NPs used, maximum
concentrations for the toxicity assessment have to be
determined for each individual particle type.
Without prior adaptation, the DCF assay would lead to
false-negative results and an underestimation of NP tox-
icity. Strikingly, in the case of CB, we also observed DCF
production in a cell-free assay possibly due to its oxidative
properties. This, in turn, leads to false-positive results and
an overestimation of the toxic effect. Therefore, our results
suggest that DCF assays used in classical toxicology
studies need to be further optimized for the use of engi-
neered NPs. Apart from lowering particle concentrations to
non-interfering levels and removing particles by washing,
we suggest testing the oxidative potential of each NP and
subsequently limiting the incubation time accordingly.
We also demonstrate interference of the engineered NPs
with the MTT assay. In particular, we found a concentra-
tion-dependent increase in MTT-formazan light absorption
for each individual NP. The contribution of CB to the
absorption of MTT-formazan is likely to result mainly
from its light absorptive capacity since we could not detect
a cell-free conversion of oxidized MTT in the presence of
CB as it has been suggested earlier (Monteiro-Riviere et al.
2009). The enhanced MTT-formazan light absorption
observed in the presence of TiO2 and of all other NPs may
be basically due to light scattering effects. Such effects
have been reported for TiO2 NPs of 40 nm size. When
applied to resin composites, these particles significantly
reduced the translucency and increased opalescence (Yu
et al. 2009). However, the contribution of NPs to the MTT
light absorption signal does not only increase with the NP
concentration used but also with the concentration of
reduced MTT-formazan present in the MTTred/MTTox
mixtures, indicating different mechanisms of interference
that cannot be predicted a priori. Therefore, determining
background signals for NPs with MTT in cell-free controls
may not be sufficient to avoid this bias in the results. Our
findings suggest that also in the MTT protocols, washing
steps should be included and NP concentrations should be
limited to non-interfering levels (10 lg cm-2 for the NP
used here) to reduce the amount of NPs present during the
measurement. However, in the case of CB, these assay
adaptations were not appropriate. Results obtained with the
optimized assay protocol indicate the interference of CB
even at particle concentrations of 10 lg cm-2. Due to its
high adsorption capacity, CB may adsorb to the surface of
the microtiter plate or the cell monolayer and may not be
efficiently removed by washing. The interference of CB
with the measurement of MTT will lead to false-negative
results and consequently to an underestimation of its
cytotoxicity. MTT assays are therefore not useful for
testing the cytotoxicity of CB. The suitability of MTT
assays to assess the in vitro toxicity of nanomaterials has
been discussed previously. Carbon nanotubes have been
demonstrated to adsorb MTT-formazan crystals, thereby
preventing solubilization (Belyanskaya et al. 2007; Casey
et al. 2007; Worle-Knirsch et al. 2006). Recently, meso-
porous silica NPs have been shown to interfere with the
MTT test by accelerating the exocytosis of formazan
crystals likely due to the perturbation of intracellular ves-
icle trafficking by silica NP uptake (Fisichella et al. 2009).
Silver nanoparticles were found to interact directly with
MTT, suggesting greater than 100 % viability of nano-
particle-exposed cells (Sadik et al. 2009). These studies
and our data demonstrate the pleiotropic ways in which
nanomaterials can interfere with the MTT assay and sug-
gest careful validation when employing this assay for NP
toxicity evaluation.
Our results also demonstrate the interference of all 24
NPs with the optical detection of reduced INT. Because
INT-formazan also absorbs in the spectrum of visible light,
the mechanisms underlying this interference might be
similar to those discussed for the NP-increased MTT
light absorption. Contrary to the interference with MTT
light absorption, however, an enhancement of INT light
absorption by NPs gives false-positive results and leads to
an overestimation of NP toxicity. Since reduced INT is
present in the supernatant of cells, NPs cannot be removed
by washing prior to the measurement. Removal of NPs by
centrifugation depends highly on the NP type and might
only be applicable when individual particles are tested.
When INT light absorption has to be measured in the
presence of NPs, the concentration of NPs should be lim-
ited to a maximum concentration that does not interfere
with the colorimetric detection. For the NPs tested in this
study, a maximum of 10 lg cm-2 was used without par-
ticle interference.
We have further determined the influence of NPs on the
enzymatic activity of LDH and observed a partial decrease
in LDH activity in the presence of ZnO NPs and fine-sized
ZnO. Since we found similar inhibition effects with ZnO
NPs and fine-sized ZnO and a similar solubility of both
ZnO particle types in cell culture medium (data not
shown), inhibition of LDH activity may be triggered by
released zinc ions as it has been described earlier for ZnCl
(Carlsson et al. 1993). In contrast to interference of NPs
with INT absorption, this enzymatic inactivation generates
false-negative results and makes ZnO NPs appear less toxic
as they actually are. This again demonstrates that even
within one single assay NP interference can influence the
toxicity outcome in different directions leading to highly
biased results and invalid conclusions.
Another confounding factor within NP toxicity screen-
ing might be the ability of NPs to specifically adsorb
proteins on their surface (Cedervall et al. 2007; Lundqvist
et al. 2008). This is particularly important when proteins
1134 Arch Toxicol (2012) 86:1123–1136
123
are used as reporters to indicate particle toxicity as it is the
case when assessing the proinflammatory potential of NPs.
A variety of NPs has been reported to adsorb proin-
flammatory cytokines. Metal oxide NPs such as TiO2 and
SiO2 seem to adsorb IL-6 (Veranth et al. 2007), and CB
was found to bind several different cytokines, for example,
GM-CSF, TGF-ß, TNFa, IL-6 and IL-8 (Brown et al. 2010;
Kim et al. 2003; Kocbach et al. 2008; Monteiro-Riviere
and Inman 2006; Seagrave et al. 2004; Val et al. 2009). In
contrast to the studies of Kocbach et al. and Brown et al.,
we could not detect any IL-8 adsorption by CB particles of
14 nm size (Brown et al. 2010; Kocbach et al. 2008).
Although Brown et al. used the same NP type as we did in
our study, their particles were suspended in PBS, whereas
we used serum-containing cell culture medium for the
preparation of NP dispersions (Schulze et al. 2008).
Kocbach et al. 2008 reported that cytokine binding could
be nearly completely reduced by adding serum proteins to
particle suspensions (Kocbach et al. 2008). Moreover,
cytokine binding has been found to increase with increas-
ing particle concentrations (Brown et al. 2010; Kocbach
et al. 2008). Thus, limiting NP concentrations to a maxi-
mum that does not display cytokine binding is of particular
importance to evade underestimating the proinflammatory
potential of engineered NPs. Our results demonstrate that
CB dispersions in serum-containing medium with particle
concentrations up to 50 lg cm-2 do not display significant
cytokine binding in cell-free controls. However, this does
not hold true for all 24 engineered NPs tested. One of the
three TiO2 NPs (TiO2 2) adsorbed considerable amounts of
cytokine when particle concentrations of 50 lg cm-2 and
below were used. As TiO2 2 differs from TiO2 1 only by
predispersion and TiO2 1 did not significantly decrease
measurable IL-8 protein levels, it can be assumed that apart
from chemical composition (Val et al. 2009) and surface
area (Brown et al. 2010), even minor modifications such as
predispersion may influence the cytokine binding proper-
ties of engineered NPs.
In conclusion, this study highlights the non-biological
artifacts obtained with classic cytotoxicity assays when
testing NPs. In particular, we observed a concentration-
dependent interference of all 24 engineered NPs with the
optical measurements used to determine the oxidative
stress (DCF), cellular metabolic activity (MTT) and cell
viability (LDH). Influence of NPs on substrates or enzymes
depended on the particle composition and, in the case of
cytokine binding, also on particle predispersion. Our results
suggest that classic cytotoxicity assays have to be further
developed, validated for each particle tested and restricted
to NP concentrations below interfering levels. Assay
adaptations have to be ascertained by a series of control
experiments for each single NP variant to obtain reliable
NP toxicity data.
Acknowledgments This work was supported by grants of the
German Federal Ministry of Education and Research (BMBF projects
NanoCare and Cell@Nano) and the state NRW (NanoPaCT). We
thank Birgit Phillip for excellent technical assistance.
Conflict of interest The authors declare that they have no conflict
of interest.
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