The cytotoxicity of cadmium-free quantum dots and
their use for cell bioimaging.
Stefaan J. Soenen†,*, Bella B. Manshian†, Tangi Aubert‡,∫, Uwe Himmelreich†, Jo Demeester┬,
Stefaan C. De Smedt┬,*, Zeger Hens‡,∫, Kevin Braeckmans∫, ┬
†
MoSAIC/Biomedical NMR Unit, Department of Medicine, Catholic University of Leuven,
Belgium; ‡Physics and Chemistry of Nanostructures, Ghent University, Belgium; ∫Center for
Nano- and Biophotonics, Ghent University, Belgium; ┬Lab General Biochemistry and Physical
Pharmacy, University of Gent, Belgium.
KEYWORDS Quantum dots, nanotoxicology, cadmium-free particles, nanoparticles.
The use of quantum dots (QDots) as bright and photostable probes for long-term fluorescence
imaging is gaining more interest. Thus far, (pre)clinical use of QDots remains limited, which is
primarily caused by the potential toxicity of QDots. Most QDots consist of Cd2+ ions, which are
known to cause high levels of toxicity. In order to overcome this problem, several strategies have
been tested, such as the generation of cadmium-free QDots. In the present study, two types of
cadmium-free QDots, composed of ZnSe/ZnS (QDotZnSe) and InP/ZnS (QDotInP) are studied with
respect to their cytotoxicity and cellular uptake in a variety of cell types. A multiparametric
cytotoxicity approach is used, where the QDots are studied with respect to cell viability,
oxidative stress, cell morphology, stem cell differentiation, and neurite outgrowth. The data
reveal slight differences in uptake levels for both types of QDots (maximal for QDotZnSe), but
1
clear differences in cytotoxicity and cell functionality effects exist, with highest toxicity for
QDotZnSe. Differences between cell types and between both types of QDots can be explained by
the intrinsic sensitivity of certain cell types and chemical composition of the QDots. At
concentrations at which no toxic effects can be observed, the functionality of the QDots for
fluorescence cell visualization is evaluated, revealing that the higher brightness of QDot ZnSe
overcomes most of the toxicity issues compared to QDot InP. Comparing the results obtained with
common Cd2+-containing QDots tested under identical conditions, the importance of particle
functionality is demonstrated, revealing that cadmium-free QDots tested in this study are not
significantly better than Cd2+-containing QDots for long-term cell imaging and more work needs
to be performed in optimizing the brightness and surface chemistry of cadmium-free QDots for
them to replace currently used Cd2+-containing QDots.
Introduction
The ongoing progress in nanotechnology is rapidly opening the doors for a wide number of
advanced biomedical applications.1-3 Novel nanosized materials (NMs), each with their own
unique properties, are generated at an incredibly fast pace, stimulating the progress of a broad
variety of biomedical research areas, including tissue engineering, neural conductance, targeted
drug delivery, multimodal whole-body imaging and combinations of both diagnostics and
therapy.4,5 Among the different types of NMs, colloidal nanoparticles (NPs) take an important
place either as (multimodal) imaging contrast agents or as therapeutic agents.6 One type of NM
that has attracted a lot of attention in particular are colloidal semiconductor quantum dots
(QDots). QDots are nanocrystals in the range of 1-10 nm diameter that are most often composed
2
of heavy metals from group 12 and group 16. The most common QDot types with the highest
quantum yields are the cadmium-containing QDots such as CdTe or CdSe. For biomedical
purposes, the QDot cores will typically be surrounded by a shell layer (e.g. ZnS) that passivates
the core and enhances the optical properties of the QDots.7 QDots possess exceptional
fluorescence properties, such as a high brightness and high photostability and broad excitation
spectra combined with very narrow, size-dependent emission spectra enabling multiplexed
imaging of cells, tissues and whole animals using a single excitation source.8,9 In contrast to
classical organic fluorophores, these properties have made QDots interesting tools for
fluorescence imaging of fixed cells and tissues where multiple markers can be analyzed
simultaneously. Additionally, QDots can be used for the continuous follow-up of single particles
over longer time periods, allowing to study the kinetics of mobile cellular markers such as cell
membrane receptors.10
The use of cadmium-containing QDots for biomedical purposes has, however, remained quite
limited due to the high toxicity of free Cd2+ ions and concerns on the potential toxic effects of
these Cd2+-containing NPs.11-13 Studies on the toxic effects of QDots have generated a lot of
disparate data, owing to, among others, differences in a) size of the QDot core, b) chemical
composition of the QDots, c) surface coating, d) presence or absence of a shell layer or e) the
experimental methodology used to evaluate QDot toxicity.14 The in vivo toxicity of Cd2+containing QDots remains largely unknown. Although there is one initial study on primates
where no toxic effects were observed under the conditions tested, this study did not take into
account any possible long-term effects.15,16 In contrast to this, most cell studies found that Cd2+containing QDots were highly toxic, although large differences exist between different cell types,
where under some conditions, particles have been described to be selectively toxic towards
3
cancer cells.17 These differences are likely due to the intrinsic differences between classical in
vitro and in vivo methods, highlighting the need for proper experimental toxicity tests.18,19 Based
on the data obtained, some general conclusions concerning Cd2+-based QDot toxicity can
however be drawn: a) the presence of a passivating shell layer typically reduces QDot toxicity, b)
a polymeric coating that envelopes the QDots typically provides the best protection in contrast to
short ligands and c) toxicity is for the most part caused by the induction of reactive oxygen
species and as a result of (intracellular) QDot degradation and release of free Cd2+ ions.11
As the presence of Cd2+ ions has been shown to be a major determinant in the toxicity of Cd2+containing QDots, several strategies have been worked out to optimize the production and
performance of Cd2+-free QDots.20,21 Originally, these QDots displayed inferior optophysical
properties compared to classical Cd2+-containing QDots, but recent advances have enabled the
generation of very bright QDots, mostly In3+-based, with high quantum yields that were stable in
aqueous media.22 These particles have demonstrated their potential use for biomedical
applications in proof-of-concept studies as Near-InfraRed (NIR) emitters and for in vitro imaging
of cancer cells or in vivo imaging of sentinel lymph nodes.22-24 However, for these materials to
develop further as biomedical research tools, next to optimizing their optophysical properties and
comparing them to Cd2+-containing QDots, their potential toxicity needs to be analyzed. Few
studies thus far have looked into the toxicity of these materials, but some promising preliminary
results have already been obtained, where toxic levels were found to be lower than for their
Cd2+-containing counterparts.23,25 The present work evaluates the cytotoxicity of two different
types of Cd2+-free QDots (ZnSe/ZnS and InP/ZnS respectively) according to a previously
established multiparametric methodology.26 This methodology enables an in-depth evaluation of
cell-NP interactions employing reproducible protocols and will provide a concentration at which
4
no toxic effects can be observed.27 As accurate determination of NP concentrations is a timeconsuming and error-prone task, a direct comparison of non-toxic NP concentrations cannot be
performed with high certainty. Therefore, at the concentration at which no toxic effects are
observed, the functionality of the QDots will be evaluated with regards to their potential for
visualization of live cells, as determined by visualizing the number of QDot-containing cells by
fluorescence microscopy during extended culture times, as described previously.12,28 Then, their
functionality at non-toxic levels can be compared directly to other NPs with similar functionality
that have been previously studied under identical conditions using the same multiparametric
methodology, such as various types of Cd2+-containing QDots or fluorescently tagged silica
NPs.12,29
Experimental procedures
Nanoparticles
Two types of Cd2+-free QDots were purchased from Mesolight LLC (Little Rock, Arkansas
USA). One type of QDots were ZnSe/ZnS core-shell QDots, the other QDots were InP/ZnS coreshell QDots. The particles were coated with 3-mercaptopropionic acid, which bestows them with
a negative surface charge. The particles were provided as 9 µM QDot stock solutions in alkaline
H2O, pH 11.
Nanoparticle characterization
Size determination by transmission electron microscopy
The size of the QDots was determined by transmission electron microscopy (TEM) using a Cs
corrected JEOL 2200 FS microscope operating at 200 kV.
5
Elemental analysis
The chemical composition of the QDots was determined by energy dispersive X-Ray
spectroscopy (EDX) using the same microscope as for the TEM observations.
Absorbance and emission spectra
Emission spectra of the QDots were recorded using a Quantifluor fluorometer (Promega,
Belgium) with an excitation wavelength of 340 nm.
Dynamic light scattering and electrophoretic mobility measurements
The hydrodynamic diameter and -potential of both types of QDots were measured using a
Nanosizer instrument (Malvern, Worcestershire, UK). For -potential measurements, the QDots
were diluted (1/500) in phosphate buffered saline (PBS: 10 mM; pH 7.0) after which the
measurements were performed (12 cycles/run) in triplicate. For determination of hydrodynamic
size, the QDots were diluted (1/500) in full culture medium (see below, section “Cell culture”)
containing 10% fetal calf serum and 5% horse serum after which the measurements were
performed (12 cycles/run) in triplicate. As a control, serum-containing media only were
measured. Upon addition of the QDots, any peaks that were shifted in size or new peaks that
emerged were considered for the calculation of the average size of the particles in order to
exclude the pure serum proteins.
QDot sterility tests
Both types of QDots were provided as sterile stock suspensions. To rule out the effect of any
biological contaminants such as endotoxins, the common endpoint chromogenic QCL-1000®
6
LAL assay (Lonza, Verviers, Belgium) was performed according to the manufacturer’s
instructions.
QDot-cell interaction studies
Cell culture
C17.2 neural progenitor cells and PC12 rat pheochromocytoma cells were cultured in high
glucose containing Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10%
fetal calf serum, 5% horse serum, 1 mM sodium pyruvate, 2 mM L-Glutamine and 1%
penicillin/streptomycin (Gibco, Invitrogen, Belgium). C17.2 cells were passaged every 48 h and
split 1/5. PC12 cells were passaged when reaching near 70% confluency and split 1/5 in tissue
culture dishes (Greiner Bio-OneBA/BV, Wemmel, Belgium) which were coated with synthetic
laminin peptide (Synthetic laminin peptide for Rat Neural Stem Cells, Millipore SA/NV,
Brussels, Belgium). To establish non-proliferating cell cultures, cells were exposed with 60 mM
Apigenin (Sigma-Aldrich, Bornem, Belgium). As particle uptake is linked with cell cycle
progression, Apigenin treatment occurred immediately after cells had been incubated with QDots
at the desired concentrations. After media removal, fresh media containing 60 mM Apigenin
were used, where media were replaced for 50% every other day with fresh Apigenin-containing
medium for the duration of the experiments.
Human umbilical vein endothelial cells (HUVECs) were maintained in endothelial
basal/growth culture medium (EBM-2/EGM-2, Clonetics, San Diego, CA) with medium changes
every 48 h. Cells were passaged when reaching near 80% confluency by lifting the cells with
0.05% trypsin (Gibco) and were plated (1/5) onto tissue-culture flasks coated with collagen. To
establish non-proliferating HUVEC cultures, cells were given endothelial cell serum-free defined
7
medium (Cell Applications, Tebu-Bio, Le Perray en Yvelines, France) immediately after cells
had been exposed to the QDots. Confluent HUVEC monolayers could then be maintained for at
least one week.
Cell-QDot studies
A detailed experimental methodology can be found in the Supporting Information that
accompanies this manuscript.
Statistical analysis
All data are expressed as mean + SEM unless indicated otherwise and analysed using one-way
analysis of variance (ANOVA). Comparisons between particle-treated groups and untreated
control groups were analysed using the Dunnett post-hoc analysis method. In all cases, the
degree of significance is indicated when appropriate (*: p < 0.05; **: p < 0.01; ***: p < 0.001).
Results and Discussion
Nanoparticle characterization
Two types of core-shell QDots were used, either InP/ZnS QDots (QDotsInP) or ZnSe/ZnS
QDots (QDotsZnSe). The chemical composition of the two types of QDots was evaluated by
elemental analysis (Supporting Figure S1), revealing that the QDots InP consist of approximately
13% InP core and 87% ZnS shell, whereas the QDotsZnSe consist of about 48% ZnSe core and
52% ZnS shell, which is in line with the descriptions given by the commercial supplier. These
QDots both had a coating of 3-mercaptopropionic acid (MPA) which results in a negative surface
charge of -32 + 2 and -27 + 2 mV for QDotsInP and QDotsZnSe, respectively. Particle diameters
8
were assessed by transmission electron microscopy (TEM; Supporting Figure S1), revealing
similar sizes for both types of QDots, being 5.9 + 0.7 nm and 5.6 + 0.6 nm for QDotsInP and
QDotsZnSe, respectively. The apparent aggregation of the QDots seen in the TEM images may be
an artifact derived from the high concentration of NPs in the stock suspension and the TEM
procedure itself which uses a vacuum system. On the other hand, the MPA coating in itself does
not provide excellent colloidal stability, which is in line with the measured hydrodynamic
diameters of the QDots exposed for 24 h in cell media (DMEM) being 115 + 12 nm for QDotsInP
and 147 + 14 nm for QDotsZnSe (Supporting Figure S2). These data reveal that the MPA coating
results in relatively stable particles at alkaline pH, where in cell culture media, some
agglomeration is seen and small clusters of nanoparticles and surface-bound molecules are
formed. The QDotsZnSe have a maximal emission at 425 nm (Supporting Figure S3B) and a
photoluminescence quantum yield (PLQY) of 36%. The QDotsInP have maximal emission at 598
nm (Supporting Figure S3A) and a PLQY of 27%. Additionally, the stock suspensions were
shown not to contain any endotoxins as evaluated by the chromogenic QCL-1000® LAL assay
(data not shown).
Cellular QDot uptake
In the present study, cell experiments were performed on three different cell types, being:
primary human umbilical vein endothelial cells (HUVEC), murine neural progenitor cells
(C17.2) and rat pheochromocytoma cells (PC12). These cells have shown to be well suited for
nanotoxicity studies12,29,30 as they have widely differing characteristics and therefore provide a
nice and general overview of nanoparticle impact on cultured cells. Additionally, the HUVEC
and C17.2 cells are frequently used in transplantation experiments for which cell labeling by
nanomaterials for cell tracking purposes is a major application.31 Furthermore, the cells have
9
been used in various studies, enabling a direct comparison of the results obtained in the current
study to those obtained in previous studies for similar particles investigated under identical
conditions.28
For biomedical applications, the particles will finally be internalized by the cultured cells,
which normally results in their enclosure in the endosomal compartments of the cell.32 Figure 1A
reveals a high level of colocalization between either type of QDot and green fluorescent protein
(GFP)-expressing endosomal compartments within the cells, revealing that both types of QDots
were indeed intraendosomally located. Based on fluorescence images, the number of distinct dots
(= QDot clusters) was counted, as described previously,12 revealing slightly but non-significantly
higher uptake levels for the QDotsZnSe in all three cell types (Figure 1B, Supporting Figure S4).
To carefully evaluate nanoparticle toxicity, it is essential to determine the number of QDots per
cell in order to link the toxicity of the particles to their functional (= intracellular)
concentration.28 As Zn2+ ions are present within the cells, the use of inductively coupled plasma
spectroscopy for determining cellular QDotZnSe levels is not that straightforward. Therefore, we
used a previously validated methodology,12,28 in which cellular QDot levels are determined. To
this end, the cells were lysed in a low molarity buffer with alkaline pH (9.8), after which the
concentration of the QDots was determined by comparing fluorescence intensity levels with the
fluorescence of known QDot stock suspensions in the same solvent. Figure1C (Supporting
Figure S4) shows the same trend as for the number of clusters per cell, where the number of
QDotsZnSe is slightly higher than the number of QDotsInP, especially in the case of the PC12 cells.
The differences in cellular uptake are somewhat surprising, given the similar hydrodynamic size
and -potential of the QDots that have an identical surface coating. This suggests that the actual
core of the particles influences “cell vision”, which is a parameter that represents how the cell
10
handles foreign materials when they are subjected to the biological environment (such as serum
components) that surrounds the cell.33 As the difference in surface charge and (hydrodynamic)
size are so minimal, and considering that both QDots consist of an outer shell of identical
chemical composition (ZnS), the difference in the chemical composition of the core seems to
play an important role. Possibly, the different core structures influence the crystal structure of the
shell layer, which in turn influences the formation of the protein corona and how these particles
will be processed by the cell. More in-depth research is needed in the future to evaluate this
hypothesis. Overall, these data suggest that both types of QDots are taken up efficiently by all
cell types and are finally located within the endosomal compartments of the cell. The QDots ZnSe
are internalized slightly more efficiently than the QDotsInP which likely reflects minor
differences in cell vision depending on the QDot core.
Effect of endosomal environment on QDot properties
One of the consequences of being located inside endosomes is that the QDots will be exposed
to low pH levels (4.5 for lysosomes; 5.5 for late endosomes). To verify the effect of this low pH
on QDot stability, both types of QDots were subjected to endosomal buffer systems of pH 7.4,
5.5 or 4.5, as described previously,12 after which the effect of the pH on their fluorescence
intensity was determined. Figure 2 reveals high optophysical stability of the particles, where
almost no decrease in fluorescence intensity could be observed over several days, even when the
QDots were exposed to the lowest pH levels. Compared to previous studies on various types of
Cd2+-containing QDots studied under identical conditions, the stability of the Cd2+-free QDots is
substantially higher, where the fluorescence intensity of Cd2+-containing QDots dropped by, on
average, 60% after 5 days exposure to pH 4.5. The higher optical stability of the Cd2+-free QDots
likely also reflects a higher chemical stability of the particles compared to the Cd 2+-containing
11
QDots. For the latter particles, it has been shown that their exposure to low endosomal pH levels
results in a degradation of the QDots themselves as a result of acid etching12,28 and subsequent
release of highly toxic Cd2+ ions in their immediate environment, increasing nanoparticle
cytotoxicity.28,34
Acute and long-term toxicity
The cytotoxic effects of both types of QDots were determined using the Alamar Blue assay for
QDot concentrations ranging from 0 to 100 nM. Figure 3A (Supporting Figure S5) shows
significant cytotoxicity starting from 60 nM for QDotsZnSe and 80 nM for QDotsInP. The
difference in toxicity between the two QDot types likely reflects the differences in cellular
nanoparticle levels, which was also slightly higher for the QDotsZnSe. However, the differences in
cellular uptake are only minimal, suggesting that other factors intrinsic to the different types of
QDots played an additional role in their cytotoxicity, such as the type of heavy metal used in the
chemical composition of the QDots. As QDot degradation results in the release of heavy metal
ions which can be highly toxic, such as Cd2+, the intracellular presence of QDots can result in a
slow but gradually increasing damage of the cells.28 To test this, cells were first labeled with
various concentrations of either type of QDot, after which they were kept in culture under nonproliferative conditions as detailed elsewhere.12 The halt in cell proliferation is essential to allow
to accurately determine the effect of QDot degradation on particle cytotoxicity without the rapid
and asymmetric dilution of cellular particle numbers which accompanies cell division.35
Furthermore, under these conditions, the cells more accurately mimic the in vivo situation, where
most cells are also not actively dividing. Figure 3B reveals no increase in cytotoxicity up to at
least 7 days after initial cell labeling. Together with the high stability of the particle fluorescence
in time, these data indicate a high chemical stability of the QDots, where environmentally-
12
induced degradation appears to be minimal. This is in stark contrast to common Cd2+-containing
QDots that have been found to degrade under endosomal pH conditions.12 This may be explained
by differences in the crystal structure of the QDots where some Cd2+-free QDots have been
described to be very robust against environmental influences such as oxidation.
Oxidative stress and secondary effects
The use of nanomaterials, such as zinc-containing NPs, is known to be closely linked to their
induction of oxidative stress, which, when it persists over longer time periods or reaches higher
levels, can result in cell death.36-39 Figure 3C (Supporting Figure S5) shows a clear induction of
reactive oxygen species (ROS), reaching significant levels at 30 nM for QDotsZnSe and 50 nM for
QDotsInP. The higher level of oxidative stress for the QDotsZnSe suggests the important role of
ROS in QDot toxicity given their higher cytotoxicity. The link between oxidative stress and
cytotoxicity is quite unclear, as all cell types have different degrees of antioxidative capacity.40
Oxidative stress can affect cell viability through several possible mechanisms, such as the
induction of mitochondrial damage, alterations in calcium fluxes, or DNA damage. Therefore,
the effect of both types of QDots on secondary oxidative stress mechanisms was investigated.
Figure 4A (Supporting Figure S6) shows no significant increase in the number of double strand
breaks for either type of QDot as revealed by phosphorylated -H2Ax staining, a common marker
for DNA double strand breaks.41 Furthermore, cellular calcium levels were slightly but not
significantly increased for both types of QDots at higher particle concentrations (Figure 4B,
Supporting Figure S7). Changes in calcium waves are important signaling mediators in cell
homeostasis, where alterations in cytoplasmic calcium waves can have drastic effects on many
important cellular processes such as cell division or cell viability.42 Calcium waves however
have only a short lifetime and die out rather fast so they can easily be overlooked in the case of
13
minor alterations.43 Therefore, the effect of the QDots on alterations in mitochondrial membrane
potential (m) was investigated as a more robust marker. Figure 4C (Supporting Figure S8)
shows a clear and significant loss of m for both types of QDots, starting from 30 nM for
QDotsZnSe and 60 nM for QDotsInP. These data are closely matched with the differences in
oxidative stress levels induced by both types of QDots as well as their cytotoxicity and therefore
suggest the importance of oxidative stress in the cytotoxic profile of both types of QDots.
The difference between QDotsZnSe and QDotsInP is somewhat strange as both types of QDots
have the same shell layer in terms of composition (ZnS). Additionally, they are coated with the
same organic coating and have a similar hydrodynamic size and -potential. Yet, similar as with
cellular uptake, the differences in the core structure of the QDots appears to affect their final
cellular effects. Thus far, the exact cause of this remains rather unclear. Given the high stability
of the particles against degradation, it is rather unlikely that many ions from the particle core will
be free in the environment to affect cell viability. Even if very low levels of degradation would
occur in time, some Zn2+ ions will be released from the shell layer in both types of QDots. For
the QDotsZnSe, more Zn2+ ions can be released from the core, elevating the total level of cellular
zinc, an essential component in cell homeostasis, above toxic thresholds. For the QDotsInP, In3+
will be released, which in itself may not reach the levels which are above the toxic threshold.
The total level of Zn2+ ions will then likely also be lower for the latter QDots, where the
contribution of In3+ and Zn2+ separately on inducing oxidative stress may be less outspoken than
higher levels of Zn2+ alone. To evaluate this in more detail, the effect of In3+ and Zn2+ ions on
cell viability (Supporting Figure S9) and oxidative stress (Supporting Figure S10) have been
investigated. These data clearly reveal that neither of the ions result in any significant
cytotoxicity at concentrations up to 12 µM (which exceeds the level that can be released from the
14
QDots), as observed for Cd2+-containing QDots.12,28 The level of cellular In3+ or Zn2+ ions were
not assessed in the current study as for Zn2+ ions, which are naturally present, determining the
level of QDot-derived Zn2+ in a cellular environment by ICP-MS is very challenging. The lack of
any toxic effects for these ions under the conditions used are in line with other reports, where, for
example, for freshwater shrimp, the LC50 of In3+ ions was found to be 60 µM compared to 0.5
µM for Cd2+.44
Interestingly however, is the induction of oxidative stress for both ions (Supporting Figure
S10). In3+ ions, which are known ROS inducers45 show a clear concentration-dependent
induction of ROS that reaches saturation levels at higher In3+ concentrations and then slowly
drop to lower levels. Zn2+ ions, on the other hand, initially do not affect cellular ROS levels up to
a concentration of 10 µM, but then result in a sharp increase at higher level. The lack of any
significant ROS inductions by low levels of Zn2+ ions is in line with their status as potent
antioxidants.46 The higher toxicity and higher induction of ROS by QDotZnSe is in apparent
contrast to these findings. However, it has also been shown that Zn2+ ions can aggravate the
cellular stress induced by other stimuli.46 In the present study, the QDots themselves may induce
oxidative stress, where low levels of free Zn2+ may further increase cellular ROS levels,
specifically in the case of QDotZnSe.
Effects of QDots on cell functionality
Nanomaterials can affect cells in many different ways, requiring a large number of parameters
to be analyzed to get a clear overview of potentially negative effects that these particles may
exert. For functionally active cells, such as HUVEC or C17.2 cells that are actively being
explored in cell transplantation studies,31 assessing cell functionality is an important aspect. It is
15
important to note that dead cells will logically not have any more functionality and therefore
only those conditions are selected at which no cytotoxicity was observed, being 60 nM for
QDotsZnSe and 80 nM for QDotsInP. In the present study, the three cell types used offer optimal
conditions for assessing important functional parameters. The widely spread HUVEC cells are
ideally suited to investigate the effect of the QDots on cell morphology and cytoskeletal
architecture.30 For QDots that are ideally suited for bioimaging applications in live cells, it is
essential that the intracellular presence of the particles does not affect cell structure. Some
particles, including QDots, have been found to disturb cell morphology and cell cytoskeleton and
some studies have shown direct effects on actin fibers.12,47 The HUVEC cells were therefore
stained for F-actin and -tubulin, after which the spreading of the cells was analyzed by confocal
microscopy and image analysis, as performed previously.43 Figure 5A shows representative
images of the cells which display a clear spreading and nice cytoskeletal architecture for
untreated control cells and cells labeled with low concentrations of QDots. Cell area distribution
was then analyzed (Figure 5B), which shows a clear decrease in cell spreading at 40 nM for
QDotsZnSe and 50 nM for QDotsInP. These effects may in part be due to the induction of ROS, but
when cells were treated with 5 mM N-acetylcysteine (NAC), a free radical scavenger that
reduced cellular ROS levels to near control values (data not shown), the decrease in cell
spreading could only be partially overcome (Figure 5C). The results demonstrate that oxidative
stress plays a major role in the cytotoxic effects of the QDots, but that for both types of particles,
other mechanisms also played an important role, such as cytoskeletal rearrangements and
reductions in cell spreading.
For C17.2 cells, cell functionality can be evaluated by determining the efficiency of cell
differentiation, where the neural progenitor cells can be differentiated into full neurons. Figure
16
5D shows a clear and significant loss in differentiation efficiency of the C17.2 cells when treated
with 50 nM QDotsZnSe, whereas QDotsInP did not appear to have any significant effect. Treating
the cells with 5 mM NAC could again only partially overcome the observed effects, suggesting
that cell differentiation was affected through other non-ROS associated mechanisms. The
differentiation of the C17.2 cells is a long process that is accompanied with high levels of cell
death, which can harden any analysis of minor effects.26 The PC12 cells present a more elegant
approach for investigating nanoparticle effects on cell functionality as they can reversibly induce
rapid neurite outgrowth that can easily be quantitated.48 This process is also accompanied with
only low levels of cell death and therefore allows picking up any effects with higher sensitivity
than the C17.2 cells. Figure 5E shows significant reductions in neurite outgrowth efficiency at 10
nM QDotsZnSe and 20 nM QDotsInP.
Taken together, these data show that at subcytotoxic concentrations, both types of QDots still
affect cell homeostasis, by impeding cell functionality and inducing oxidative stress. To evaluate
the functionality of the particles themselves, further tests are performed at the concentrations of
the particles at which no adverse effects were observed, being 10 nM for QDotsZnSe and 20 nM
for QDotsInP. When compared to different types of Cd2+-containing core-shell QDots studied
under identical conditions, these concentrations are approximately 10-fold higher (ranging from
0.5 to 2 nM).12,28 This high level of particle concentrations that can be tolerated by the cells is
likely caused by a combination of several factors, in part being the lower intrinsic cytotoxicity of
both types of Cd2+-free QDots, which is in line with other studies reporting lower toxicity levels
for Cd2+-free QDots.25 However, in order to accurately assess particle toxicity, the intrinsic
toxicity of the particles must be considered. A direct comparison of the different particles is
made impossible by differences in their size, surface coating and the associated changes in
17
cellular interaction such as alterations in cellular uptake levels.28 Furthermore, a comparison of
particle toxicity based on concentrations is prone to big errors as accurate determination of
nanoparticle numbers in stock suspensions is extremely tedious.14
Functionality of the QDots for cell imaging
Based on the determination of the safe concentrations of both types of QDots, their functionality
can be assessed by evaluating how long they can be used for visualizing live, actively dividing
cells, which would be one major application of the QDots. This then allows a much better
comparison of different nanoparticles with the same functionality as it includes a large number of
potential differences between particles such as differences in uptake levels, errors in determining
stock concentrations or QDot PLQY.28 In practice, cells are seeded with either type of QDot at
non-cytotoxic concentrations for 24 h (being 10 nM for QDotsZnSe and 20 nM for QDotsInP), after
which cells are kept in culture. Upon every two cell divisions, cells are reseeded at their initial
density and part of the cell population is kept for microscopy analysis, as described in detail in
the Supporting Experimental Methodology. The reseeding of the cells is performed in order to
avoid the need for low cell densities at initial time points (HUVEC and PC12 cell proliferation
requires optimal cell densities) and to avoid cell cultures to become overpopulated which would
impede cell division due to contact inhibition. Figure 6 shows that the QDotsZnSe can be used to
visualize cells for approximately 4 cell cycles, whereas the QDots InP allow cells to be tracked for
approximately 6 cell cycles. For various types of core-shell Cd2+-containing QDots, cells could
be tracked for approximately 2 to 4 cell cycles, indicating that the Cd2+-free QDots are
approximately 1.5-fold better than some Cd2+-containing QDots previously tested.12,28 The
difference in particle functionality is therefore far less than expected based on the concentrations
of the particles only. This is likely caused by the relatively low brightness of the Cd2+-free QDots
18
compared to some Cd2+-containing QDots. Additionally, this may be due to a lower
concentration of QDots in the stock suspension than originally determined as well as the short
organic ligands which may result in lower colloidal stability of the particles in cell medium and
reduced cellular uptake.49 Overall, the Cd2+-free QDots appear to be well suited alternatives for
Cd2+-containing QDots for cell bioimaging purposes, where the QDotsZnSe appear to be slightly
less suited than the QDotsInP, likely because of a higher intrinsic toxicity and lower brightness of
the QDotsZnSe. This is further supported upon determining the cellular fluorescence intensity of
the QDot-labeled cells, where QDotZnSe-labeled cells have a much lower fluorescence intensity
than the QDotInP-labeled cells (Supporting Figure S11). At this stage, however, both types of
QDots appear to be similar to common Cd2+-containing QDots. This is further supported by a
recent study, where more advanced Cd2+-containing QDots (i.e. so-called gradient alloy QDots,
with identical surface coating to the QDots evaluated here) that were found to have a PLQY of
up to 90%, resulted in a similar duration of cell tracking at non-toxic conditions (up to 6 cell
doublings). Therefore, the Cd2+-free QDots examined in the current study appear to be similar to
more advanced Cd2+-containing QDots. In the future, further improvements in surface chemistry
and PLQY of the Cd2+-free QDots may boost their functionality to higher levels, especially for
the QDotsInP. At this stage, however, more research needs to be performed for these particles to
be optimized and to reach their full potential in replacing current Cd2+-containing QDots.
Conclusion
The present work shows that the cadmium-free QDots, despite having a high chemical stability
and low intrinsic toxicity, appear to only be slightly better than commonly used Cd 2+-containing
19
QDots in terms of cell visualization. This is primarily due to the relatively low PLQY and
associated lower brightness of both types of cadmium-free QDots tested in this study. Although
the QDotInP and QDotZnSe both possess a ZnS shell layer, are of similar size and have the same
surface chemistry, they result in slight differences in cellular uptake levels, but significant
differences in toxic effects, which are most pronounced for QDotZnSe. These data indicate the
importance of the chemical composition of the QDot core in their final interactions with
biological entities such as cells or whole organisms. The use of the multiparametric methodology
enables to define non-toxic particle concentrations and sheds some insight into the mechanisms
involved in the negative effects of the QDots. Additionally, using the same methods and cell
types and investigating particle toxicity under well-defined and controllable conditions allows to
compare the results obtained with results for other particles studied in the same way. This
comparison reveals that cells can tolerate a higher level of cadmium-free QDots than cadmiumcontaining QDots, but when taking particle brightness and uptake levels into account, similar
results are obtained, where cadmium-free QDots are only moderately more functional. This
comparison shows the importance of particle functionality when assessing particle toxicity as
comparing particle concentrations does not give an appropriate estimation of the true differences
in particle toxicity, as many parameters such as errors in determining particle stock
concentration, cellular uptake levels etc are not taken into account. The low intrinsic toxicity of
the cadmium-free QDots offers a lot of potential for future applications, where more stable
coatings, such as functionalized silica shells could be applied to optimize colloidal stability and
particle brightness in aqueous solvents while maximizing cellular uptake.
Figures
20
Figure 1. Cellular uptake of QDots. (A) Representative confocal images of HUVEC cells
transiently expressing Lamp-1-GFP (marker for late endosomes and lysosomes; green channel)
incubated with 80 nM QDotInP (top row; red channel) or QDotZnSe (bottom row; blue channel) for
24 h. A merged image of both the QDots and the GFP-expressing endosomes is shown in the
right column. The area indicated by the white rectangle in the merged images is shown in high
resolution on the right. Scale bars: 40 µm. (B) The number of QDot clusters per cell as quantified
for 30 cells. (C) The total number of QDots per HUVEC cell as a function of the QDot
concentration for both QDotInP (light gray)- and QDotZnSe (dark gray) as quantified by measuring
total fluorescence intensity levels. Data are shown as mean + SEM (n = 4).
21
Figure 2. The fluorescence intensity levels of QDotInP and QDotZnSe exposed to buffer systems
of different pH values, being 7.4 (cytoplasmic); 5.5 (late endosomes) or 4.5 (lysosomes) for up to
5 days. Fluorescence intensity levels were measured every day. Data are presented as values
relative to the fluorescence intensity of QDots exposed to pH 7.4 and measured immediately.
Data are expressed as mean + SEM (n = 3).
22
Figure 3. (A) Viability of HUVEC as a function of QDot concentration (ranging from 20 to 100
nM) for both QDotInP (light gray) and QDotZnSe (dark gray) after 24 h incubation. (B) Viability of
non-proliferating HUVEC cells as a function of both QDot concentration (40, 60 and 80 nM) and
time (1-7 days post-QDot incubation). (C) ROS levels of HUVEC cells exposed to varying
concentrations (10-60 nM) of QDotInP (light gray) or QDotZnSe (dark gray). Data are represented
as mean + SEM (n = 4) and expressed as relative to untreated control cells. The degree of
significance between samples and controls is indicated when appropriate (*: p < 0.05; **: p <
0.01; ***: p < 0.001). Statistical significance between QDotInP and QDotZnSe samples is indicated
by #.
23
Figure 4. Secondary ROS effects of Cd2+-free QDots. (A) The level of phosphorylated -H2Ax,
(B) the level of cellular calcium or (C) mitochondrial viability in HUVEC cells as a function of
QDot concentration (ranging from 10 to 60 nM) for both QDotInP (light gray) and QDotZnSe (dark
gray) after 24 h incubation. Data are expressed as mean + SEM (n = 3). The degree of
significance between samples and controls is indicated when appropriate (*: p < 0.05; **: p <
0.01; ***: p < 0.001). Statistical significance between QDotInP and QDotZnSe samples is indicated
by #.
24
Figure 5. (A) Representative fluorescence images of HUVEC cells either untreated (top row) or
exposed to 20 nM QDotZnSe (second row), 50 nM QDotZnSe (third row), 20 nM QDotInP (fourth
row) or 50 nM QDotInP (bottom row) for 24 h. Cells have been stained for F-actin (red, left
column) and -tubulin (green, middle column). The right column shows a merged image of both
the green and red channel. Scale bars: 150 µm. (B) The average cell area of HUVEC cells as a
function of QDot concentration (ranging from 0 to 60 nM) for both QDot InP (light gray) and
QDotZnSe (dark gray) after 24 h incubation. (C) The average cell area of HUVEC cells as a
25
function of QDot concentration (ranging from 0 to 60 nM) upon 24 h incubation with either
QDotInP (light gray) or QDotZnSe (dark gray) in the presence of 5 mM NAC, a free radical
scavenger. (D) The level of C17.2 cells that differentiated into full neurons upon 1 week
exposure to neuronal induction media as a function of QDot concentration (ranging from 0 to 60
nM) for both QDotInP (light gray) and QDotZnSe (dark gray) after 24 h incubation. (E) The level
of PC12 neurite outgrowth upon 2 days exposure to nerve growth factor as a function of QDot
concentration (ranging from 0 to 60 nM) for both QDot InP (light gray) and QDotZnSe (dark gray)
after 24 h incubation. (B-E) Data are expressed as mean + SEM (n = 4). The degree of
significance between samples and controls is indicated when appropriate (*: p < 0.05; **: p <
0.01; ***: p < 0.001). Statistical significance between QDotInP and QDotZnSe samples is indicated
by #.
26
Figure 6. (A) Representative fluorescence images of HUVEC cells exposed to non-cytotoxic
concentrations of QDotInP (20 nM; top row) or QDotZnSe (10 nM; bottom row) for 24 h, after
which the cells were kept in culture. The images shown are taken immediately after QDot
exposure (D0) or after 2 (D2), 4 (D4) or 6 (D6) average cell doubling times. The images are
merged images of the fluorescent QDots (QDotInP: red; QDotZnSe: blue) and phase contrast
images of the cells. Scale bars: 150 µm. (B-D) The percentage of (B) C17.2, (C) HUVEC or (D)
PC12 cells containing QDotsInP (light gray) or QDotZnSe (dark gray) upon further culture of preincubated cells. The data are expressed as a function of the number of cell divisions the cells
underwent post-incubation. Data are given as mean + SEM (n = 4). The horizontal line indicates
27
50% of QDot positive cells, which is seen as the cut-off value below which the ability of the
QDots to visualize cells is referred to as insufficient.
ASSOCIATED CONTENT
Supporting Information. Elemental analysis and transmission electron micrographs; dynamic
light scattering plots of the nanoparticles used; quantum dot emission spectra; cellular uptake of
the quantum dots in C17.2 and PC12 cells; effects of the quantum dots on viability, ROS, DNA
damage, calcium levels and mitochondrial health in C17.2 and PC12 cells and a full
experimental methodology. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*(S. J. S.) Email: Stefaan.Soenen@med.kuleuven.be
*(S. C. D.) Email: Stefaan.DeSmedt@ugent.be
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
Funding Sources
S. J. S. is a postdoctoral fellow from FWO-Vlaanderen. The authors acknowledge FWOVlaanderen for financial support.
28
Abbreviations
DLS: dynamic light scattering
DMEM: Dulbecco’s modified Eagle’s medium
EDX: energy dispersive X-Ray spectroscopy
HUVEC: human umbilical vein endothelial cell
GFP: green fluorescent protein
MPA: 3-mercaptopropionic acid
NAC: N-acetylcysteine
NIR: near-infrared
NMs: nanomaterials
NPs: nanoparticles
PBS: phosphate buffered saline
PLQY: photoluminescence quantum yield
QDots: quantum dots
QDotZnSe: ZnSe/ZnS quantum dots
QDotInP: InP/ZnS quantum dots
ROS: reactive oxygen species
29
SEM: standard error to the mean
TEM: transmission electron microscopy
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