Copyright © 2012 American Scientific Publishers
All rights reserved
Printed in the United States of America
Journal of
Nanoscience and Nanotechnology
Vol. 12, 2160–2168, 2012
Cyto-/Genotoxic Effect of CdSe/ZnS Quantum Dots in
Human Lung Adenocarcinoma Cells for Potential
Photodynamic UV Therapy Applications
Young Joo Choi1 , Yang Jee Kim2 , Joong Won Lee1 , Younghyun Lee1 ,
Yong-beom Lim3 , and Hai Won Chung1 ∗
1
School of Public Health and Institute of Health and Environment, Seoul National University, 1 Gwanak-ro,
Gwanak-gu, Seoul, 151-742, Republic of Korea
2
Chung-Ang University, Heukseok-dong, Dongjak-gu, Seoul, 156-756, Republic of Korea
3
Translational Research Center for Protein Function Control & Department of Materials Science and Engineering,
Yonsei University,
Seoul,by
120-749,
Republic
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Yonsei University
RESEARCH ARTICLE
Quantum dots (QDs) are luminescentIPnanoparticles
(NPs) with promising potential in numerous
: 165.132.61.49
medical applications, but there remain
persistent
human
health and safety concerns. Although
Fri, 31 Aug 2012 02:48:17
the cytotoxic effects of QDs have been extensively investigated, their genotoxic effects remain
under-explored. This study scrutinized the cyto- and genotoxic effects of QDs with a Cadmium
selenide/Zinc sulfide (CdSe/ZnS) core/shell, and suggests comprehensive guidelines for the application of QDs in cancer therapy. QDs were used to treat A549 cells in the presence and
absence of ultraviolet A/B (UVA/UVB) irradiation. QD-induced cell death was evaluated by 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), apoptosis, and lactate dehydrogenase
(LDH) assays, as well as by real-time PCR analysis of differential mRNA levels of genes, such
as ataxia telangiectasia mutated (ATM), p53, and caspase-9, involved in apoptosis. The genotoxic
effect of CdSe/ZnS QDs was measured in human cancer cells, for the first time, by comet and
micronucleus assays. Treatment with CdSe/ZnS QDs and UVB irradiation resulted in the most
severe extent of cell death, indicating strong induction of phototoxicity by CdSe/ZnS QDs in the
presence of UVB. Both apoptotic and necrotic cell death were observed upon QDs and UVB combined treatment. The induction of Olive tail moments and micronuclei formation was also most
significant when CdSe/ZnS QDs and UVB irradiation were combined. Our results on the genotoxic
effect and mechanistic details of CdSe/ZnS QD-induced cell death suggest that UVB irradiation is
the most effective method for increasing the potency of QDs during photodynamic cancer therapy.
Keywords: CdSe/ZnS QDs, UVA/UVB, Apoptosis, Necrosis, ROS, DNA Damage.
1. INTRODUCTION
Nanomedicine has become one of the most promising
and attractive fields of nanotechnology.1–3 Nanometersized materials have unique and valuable properties, compared with conventional bulk materials, and numerous
advantages accompany nano-scale biomaterials, which typically range in size from tens to hundreds of nanometers. Nanotechnology-based photodynamic therapy (PDT),
one of the most promising cancer-treatment nanotechnologies, is noninvasive and fairly safe for repeated
∗
Author to whom correspondence should be addressed.
2160
J. Nanosci. Nanotechnol. 2012, Vol. 12, No. 3
administration.4 Moreover, a tumor-specific targeting strategy could be developed with appropriate functionalization
of nanoparticles (NPs).
PDT uses special drugs, called photosensitizing agents,
and visible or near-infrared illumination at specific wavelengths to kill cancer cells. Reactive oxygen intermediates
are generated during photoactivation of a photosensitizer
(PS) drug, and these immediately react with and damage vital biomolecules in cell organelles, causing cell
death.5 PDT was first used to treat cancer more than
100 years ago; however, in the earlier days, the administration of porphyrins and phthalocyanines induced severe
side effects attributable to non-specific drug delivery and
photoactivation.6
1533-4880/2012/12/2160/009
doi:10.1166/jnn.2012.5781
Choi et al.
Cyto-/Genotoxic Effect of CdSe/ZnS Quantum Dots
J. Nanosci. Nanotechnol. 12, 2160–2168, 2012
2161
RESEARCH ARTICLE
Quantum dots (QDs), which are inorganic semiconduc2. MATERIAL AND METHODS
tor nanocrystals, have become one of the most attrac2.1. Cell Culture
tive nanomaterials for bioimaging and cancer therapy.
The development of QD technology has paved the way
Adenocarcinoma human alveolar basal epithelial cells
for using QDs as agents in medical diagnostics, cancer
(A549 cells) were obtained from the Korean Cell Line
diagnosis, and PDT for cancer. QD technology has been
Bank (Seoul, Korea) and cultured in RPMI 1640 suppleapplied in many types of animal models such as mouse
mented with filtered 10% fetal bovine serum, penicillin,
and pig models.7 8 The most salient material properties
and streptomycin (100 U/mL of each), at 37 C in a
of QDs include their bright fluorescence emission, excephumidified atmosphere of 5% CO2 . CdSe/ZnS quantum
tional photostability, large surface area, and large twodot (Lumidot™ ) was purchased from Sigma-Aldrich.
photon absorption cross-section.9–12
Previous studies have shown that QDs with a Cadmium
2.2. Preparation and Characterization of
selenide (CdSe) core induce cell death by increasing
CdSe/ZnS QDs
reactive oxygen species (ROS) and inhibiting survivalCdSe/ZnS QDs dispersed in toluene were supplied by
related signaling events.13 QDs also induce apoptosis via
Sigma-Aldrich (St. Louis, MO, USA). Toluene was
mitochondrial-dependent pathways involving Fas upregremoved via rotary evaporation (Rotavapor R-210; Buchi
ulation and lipid peroxidation in human neuroblastoma
Laboratory Equipment, Zurich, Switzerland), and the QDs
cells.14 15 The cytotoxicity of CdSe-core QDs is correlated
were suspended in distilled water. This solution was soniwith the release of free Cd2+ . One of the most efficient
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30 min at 4 C before each experiment. Dynamic
ways of reducing QD toxicity is to cover the CdSe core
Yonsei University
light scattering (DLS) is an indirect method for measurwith a Zinc sulfide (ZnS) coating layer. In the present
IP : 165.132.61.49
ing particle size. Briefly, the fluctuations in scattered light
study, the ZnS shell plays an important roleFri,
as a31barrier
Aug 2012
02:48:17
intensity,
which are caused by Brownian motion of susto prevent CdSe from contacting the surrounding solvent,
pended
particles,
are measured over time. DLS was meathus protecting the CdSe core against dissolution through
sured
at
room
temperature
with an ALV/CGS-3 Compact
ionization. The ZnS shell also increases the QD quantum
14
Goninometer
System
(Hessen,
Germany) equipped with an
yield.
He-Ne
laser
operating
at
632.8
nm. The scattering angle
Despite the widespread use of QDs in diverse biomed
was
90
.
The
size
distribution
was determined using a
ical fields, including imaging, diagnosis, and PDT, a
constrained
regularization
method.
Transmission electron
detailed picture of the cellular effects of Cadmium
microscopy
(TEM;
100CX;
JEOL,
Tokyo,
Japan) was used
selenide/Zinc sulfide (CdSe/ZnS) QDs has not been estabto
determine
the
size
and
shape
of
the
CdSe/ZnS
QDs.
lished. Here, we investigated the cyto-/genotoxic effects
Absorption
peaks
of
the
CdSe/ZnS
QDs
were
measured
and mechanistic details of the cell death pathway induced
using an ultraviolet-visible (UV-Vis) spectrophotometer
in human lung adenocarcinoma cells (A549 cells) by
(V-650; Jasco, Tokyo, Japan), and photoluminescence
CdSe/ZnS QDs in the presence and absence of UV irraspectra were detected with a fluorescence spectrophodiation. A549 cells are commonly used in cancer studies,
tometer (F-4500; Hitachi, Tokyo, Japan). The QD surincluding PDT studies.16–18 Lung cancer is the leadface charge was measured in distilled water using a
ing cause of cancer deaths, with a cure rate of only
zeta potential analyzer (Zeta plus; Brookhaven Instru13% and patients do not survive 2 years owing to late
ments Corp., Holtsville, NY, USA). Scanning electron
detection.19 20 It is reported that engineered nanoparticles,
microscopy (SEM), with an energy dispersive X-ray
such as silver nanoparticles (Ag NP), nano titanium diox(EDX) spectrometer as a detector (AXS; Bruker, Bad Heride (TiO2 ), and carbon nanotube (CNT), can cause lung
renalb, Germany), was used to identify the major eledamage via enhanced inflammatory responses, lung funcment types in A549 cells. Cells were counterstained with
tion change, and fibrosis of lung tissue, finally inducing
DAPI (4, 6-diamidino-2-phenylindole)-containing mountlung cancer.21–23
ing medium to visualize cellular uptake of CdSe/ZnS
To determine the conditions in which QDs can be most
QDs by fluorescence microscopy (Vectorshield, Peterboreffective as a PDT photosensitizer, we tested the cytotoxough, UK). Confocal luminescence images of A549 cells
icity of CdSe core/ZnS shell QDs with and without UV
were obtained using a confocal microscope (Carl Zeiss,
irradiation in A549 cells using MTT, lactate dehydrogeOberkochen, Germany) equipped with a 405-nm diode
nase (LDH), and apoptosis assays.
laser and 488-nm Ar laser.
The genotoxicity induced by CdSe/ZnS QDs in human
lung cancer cells was measured primarily by micronucleus
2.3. QD Cytotoxicity
and comet assays. Furthermore, we analyzed differential
mRNA levels of genes involved in apoptosis, to investigate
The cytotoxic effects induced by the CdSe/ZnS QDs
the global effect of CdSe/ZnS QDs and UV irradiation on
in A549 cells were evaluated by MTT (3-[4,5dimethylthiazol-2-yl] 2,5-diphenyl-tetrazolium bromide)
A549 cells.
Cyto-/Genotoxic Effect of CdSe/ZnS Quantum Dots
assay. A549 cells were seeded in a 96-well plate at a density of 10,000 cells per well and incubated for 24 h at
37 C in a humidified atmosphere of 5% CO2 . Then, the
cells were treated with CdSe/ZnS QDs at different concentrations with/without UVA or UVB irradiation. After
a 24-h incubation, the cells were treated with 0.5 mg/mL
MTT solution (formazan powder; Sigma Aldrich), and
the plate was incubated for 4 h. The violet formazan
crystals were dissolved in DMSO and the absorbance at
560 nm was measured using a microplate reader (Tecan,
Männedorf, Switzerland).
Choi et al.
2.7. Measurement of ROS
The level of intracellular reactive oxygen species (ROS)
generation was determined using 5-(and-6)-carboxy-2 ,
7 -dichlorofluorescein diacetate (DCFDA; Molecular
Probes, Eugene, OR, USA). A549 cells were treated with
QDs for 2 h, and then they were irradiated with UVA
or UVB. After 1 h, the samples were centrifuged and
added with new medium containing 20 M DCFDA in
the dark. The cells were incubated for 20 min at 37 C
and monitored using a fluorescence spectrophotometer at
535 nm emission with an excitation wavelength of 485 nm
(Tecan, Männedorf, Switzerland).27
2.4. UVA/UVB Irradiation
2.8. Measurement of Apoptosis and Necrosis
Cells were plated in growth medium in a 96-well plate
and exposed to UVA (2 J/cm2 or UVB (0.3 J/cm2 light,
Apoptosis induced by CdSe/ZnS QDs with or without
using a Bio-Link BLX irradiation system (Vilber-Lourmat,
UVA or UVB irradiation was assessed using a termiMarne-la-Vallee Cdex 1, France) equipped with five 8 W
nal deoxynucleotidyl transferase dUTP nick end labelUV lamps (UV-A Vilber-Lourmat T-8L with peak irradiing apoptosis
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to: assay (Cell Death Detection ELISAPLUS kit;
ance at 365; UV-B Vilber-Lourmat T-8M with peak irraRoche Applied Science, Mannheim, Germany), accordYonsei University
diance at 312 nm). Cell viability was determined by MTT
ing to the manufacturers’ protocol. Briefly, A549 cells
IP : 165.132.61.49
assay.
were treated with CdSe/ZnS QDs, followed by UVA or
Fri, 31 Aug 2012 02:48:17
RESEARCH ARTICLE
2.5. Single Cell Gel Electrophoresis Assay
Single-cell gel electrophoresis was performed as described
previously by Singh et al.24 A549 cells were treated with
CdSe/ZnS QDs for 3 h, followed by UVA (2 J/cm2 ) or
UVB (0.3 J/cm2 ) exposure. After being washed twice with
phosphate-buffered saline (PBS), the cells were maintained
at 4 C to prevent DNA repair. Slides were prepared
according to the method of Singh.24 Briefly, images of 60
cells randomly selected from each sample were analyzed,
using a Komet 5.5 image analysis system (Kinetic Imaging
Ltd., Nottingham, UK). The Olive tail moment (OTM) of
each cell was measured under a fluorescence microscope
(Nikon, Tokyo, Japan) equipped with a 515- to 560 nm
excitation filter and 590 nm barrier filter.
UVB irradiation. After 24 h, the culture supernatant was
removed, and the cells were incubated with lysis buffer.
After centrifugation, an aliquot of the supernatant was
transferred to a streptavidin-coated microplate well and
incubated with anti-histone and anti-DNA antibodies for
2 h at room temperature, to bind nucleosomes in the supernatant. The immobilized immunocomplexes were washed
three times to remove cell components and were detected
by incubation with peroxidase substrate. Absorbance at
405 nm was measured using a microplate reader.
Necrosis was determined by measuring LDH released
into the medium by necrotic cells upon plasma membrane
rupture (Cytotoxicity Detection KitPLUS , Roche Applied
Science). Cells were cultured as for the apoptosis assay.
Briefly, substrate reagent was incubated with the culture
supernatant, and the absorbance at 490 nm was read using
a microplate reader.
2.6. Cytokinesis-Block Micronucleus Assay
The cytokinesis-block micronucleus (MN) assay was performed as described by Fenech.25 A549 cells were cultured in RPMI 1640 medium supplemented with 10%
fetal bovine serum, incubated with CdSe/ZnS QDs for
3 h, and then irradiated with UVA or UVB. Cytochalasin B (4 g/mL; Sigma-Aldrich) was added 20 h after
the start of culture, and the incubation was continued
for an additional 28 h. After a total incubation period
of 48 h, the cells were harvested, treated with hypotonic
0.075 M KCl solution for 1 min, and washed twice with
fixative solution (methanol:acetic acid, 3:1). Air-dried cell
preparations were stained with Giemsa solution (5%). In
total, 1,000 binucleated cells with well-preserved cytoplasm were scored according to standard criteria.26
2162
2.9. RNA Extraction and Quantitative Real
Time-Polymerase Chain Reaction (PCR)
Total RNA was extracted from A549 cells treated with
CdSe/ZnS QDs in the presence or absence of UVA or UVB
irradiation, using an AxyPrep Multisource Total RNA
Miniprep kit (Axygen Biosciences, Union City, CA, USA)
according to the manufacturer’s instructions. For cDNA
synthesis, total RNA was reverse transcribed using a random primer and a reverse transcription system (Promega,
Madison, WI, USA). Real-time PCR was performed using
an Applied Biosystems 7300 real-time PCR system (Foster
City, CA, USA) and Express Start SYBR Green qPCR
Supermix (Invitrogen, Burlington, ON, Canada). The synthesized primer sequences for the reactions are shown
J. Nanosci. Nanotechnol. 12, 2160–2168, 2012
Choi et al.
Cyto-/Genotoxic Effect of CdSe/ZnS Quantum Dots
Table I. Primer sequences used for quantitative real-time PCR.
Sequence (5 -3 )
Primer pair
ATM
p53
caspase-9
GAPDH (endogenous control)
-CGAGGTGAGCGGATCACAA-; -TTGGCCCACAGCAACCTT-GTGAGCGCTTCGAGATGTTC-; -ATGGCGGGAGGTAGACTGAC-GCTCTTCCTTTGTTCATCTCC-; -CATCTGGCTCGGGGTTACTGC–
-GGAAGGTGAAGGTCGGAGTCA-; -GTCATTGATGGCAACAATATCCACT-
showed a potential of +4058 ± 797 mV, confirming the
colloidal stability of CdSe/ZnS QDs. The shape and size
of the QDs were also analyzed by TEM, which revealed
that the diameter of individual CdSe/ZnS QD nanoparticles in the aggregates is approximately 4 nm (Fig. 2).
The CdSe/ZnS QD particles internalized after a 4-h cell
treatment were examined by EDX spectroscopy (Fig. 3).
Spectral peaks demonstrated the presence of Cd, Se, Zn,
and S. Cellular uptake was further confirmed by confocal
2.10. Statistical Analysis
microscopy, which also showed the intracellular localizaDelivered by Ingenta
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tion of CdSe/ZnS QDs in A549 cells. Cells were treated
The effects of CdSe/ZnS QDs with or without
UVA University
Yonsei
with 8.3 g/mL QDs, and confocal Z-stack analysis of
or UVB irradiation on DNA were analyzed using
IP :non165.132.61.49
the 02:48:17
treated cells verified the intracellular translocation of
parametric Kruskal–Wallis one-way analysis
of
variFri, 31 Aug 2012
ance (ANOVA) and the Mann–Whitney U test. One-way
CdSe/ZnS QDs (Fig. 4).
ANOVA followed by Dunnett’s or Tukey’s test was applied
to analyze cell viability, ROS generation, induction of
apoptosis and necrosis, and mRNA expression, using SPSS
software v. 12 (SPSS Inc., Chicago, IL, USA). Linear
(A)
trends for the induction of apoptosis and necrosis were
tested using a linear regression model.
in Table I. The thermal cycling conditions were incubation
at 94 C for 5 min, followed by 40 cycles of 94 C for
15 s, 55 C for 30 s, and 72 C for 30 s. Each assay was
performed in triplicate, with GAPDH as an endogenous
control for normalizing expression levels.
Data are expressed as the ratios of ataxia telangiectasia
mutated (ATM), p53, and caspase-9 to GAPDH, according
to the comparative threshold cycle method (2−Ct ).
3.1. Physicochemical Characterization and
Intracellular Distribution of QDs
Core–shell type CdSe/ZnS QDs (particle size, 3.3 nm; em ,
530 nm) was obtained from the supplier as a toluene solution. Toluene was removed by rotary evaporator to complete dryness. Then the QDs were suspended in distilled
water, and the physicochemical and photophysical properties of aqueous QDs were analyzed by DLS, UV-Vis spectroscopy, steady-state fluorescence emission spectroscopy,
zeta potential, and TEM. The average size of CdSe/ZnS
QDs was 105 ± 1555 nm as determined by DLS, indicating that QD particles are aggregated together in aqueous
environment. However, the size of QD aggregates is still
in the nano-regime, which is small enough for efficient
endocytic entry into cells. The UV-Vis absorption spectrum of CdSe/ZnS QDs showed broad continuous absorption at wavelengths extending from the UV to the visible
range (Fig. 1(A)). The photoluminescence emission spectrum was narrow, with maximum emission at 535 nm
(Fig. 1(B)). These results indicate that photophysical properties of the QDs are largely preserved in aqueous environment. The zeta potential measurement in distilled water
J. Nanosci. Nanotechnol. 12, 2160–2168, 2012
(B)
Fig. 1. Characterization of CdSe/ZnS quantum dots (QDs). UV-Vis
absorption spectrum of QDs (A). Photoluminescence (PL) spectrum
(emission wavelength = 535 nm) of QDs. The maximum emission
occurred at a wavelength of 535 nm (B).
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RESEARCH ARTICLE
3. RESULTS
Cyto-/Genotoxic Effect of CdSe/ZnS Quantum Dots
Choi et al.
Fig. 4. Confocal images of internalized quantum dots (QDs) in
A549 cells. Magnification: ×630. 3-D Z-stack image shows the localizaFig. 2. Image of transmission electron microscope of QDs. Scale bar is
tion of the QDs in A549 cells. Green, QDs; blue, cell nuclei stained with
50 nm. Magnification: × 400,000. The inset shows a higher
resolution.
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DAPI. Arrows indicate the QDs.
Agglomerates of QDs are observed.
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3.3.02:48:17
Assessment of Apoptosis and Necrosis
Fri, 31 Aug 2012
RESEARCH ARTICLE
3.2. Phototoxicity of QDs in A549 Cells under UV
Irradiation
MTT assays were performed in A549 cells to assess
CdSe/ZnS QD cytotoxicity with and without UVA or UVB
irradiation. An overall dose-dependent decrease in cell viability was observed. The effect of CdSe/ZnS QD photoactivation on cell viability was more significant when
the cells were irradiated with UVB than with UVA irradiation (Fig. 5). The A549 cells were more susceptible to the effect of CdSe/ZnS QDs combined with UVB
irradiation (68.81% cell viability) than to the effects of
CdSe/ZnS QDs plus UVA irradiation (82.01% cell viability) or CdSe/ZnS QDs (1.6 g/mL) alone (89.2% cell
viability).
Fig. 3. Energy dispersive X-ray (EDX) specta of CdSe/ZnS quantum
dots. Confirming the presence of Cd, Se, Zn, and S. Measuring time:
5 min. High voltage: 10 keV.
2164
We next conducted apoptosis assays to investigate the
mode of CdSe/ZnS QD-induced cell death in the presence
or absence of UVA or UVB irradiation. We used a one-step
sandwich ELISA assay in which mouse monoclonal antibodies directed against DNA and histones immunoreact
with mono- and oligonucleosomes in the cytoplasmic fractions of cell lysates to colorimetrically quantify the relative
level of apoptosis. This assay showed that CdSe/ZnS QDs
Fig. 5. Cell viability assay for quantum dots (QDs) in the presence
and absence of ultraviolet (UV) light. A549 cells were incubated in a
96-well microplate for 24 h and then treated with QDs with and without UVA/UVB. Cell viability was determined by MTT assay. Data are
percentages of control, and error bars represent standard errors from
triplicate experiments. Results were statistically analyzed with Dunnett’s
post-hoc test (∗∗p < 001 compared with control). Letters denote significant differences among treatments using Tukey’s HSD test after one-way
ANOVA (p < 005).
J. Nanosci. Nanotechnol. 12, 2160–2168, 2012
Choi et al.
Cyto-/Genotoxic Effect of CdSe/ZnS Quantum Dots
induced A549 cell apoptosis in a dose-dependent manner
(Fig. 6(A)), with a more significant increase in apoptosis
generally observed in cells treated with both CdSe/ZnS
QDs and UVB. Interestingly, the induction of apoptosis
in the UVB-treated group was somewhat reduced at the
highest QD concentration (16.6 g/mL).
In contrast, the LDH assay, which is a sensitive detection method for necrotic cell death, revealed significant
and consistent increases in LDH release in proportion
to increases in QD concentration (Fig. 6(B)). Taken
together, the results indicate that CdSe/ZnS QDs at relatively low concentrations induced apoptosis, but QDs at
(A)
higher concentrations had a tendency to induce necrosis, rather than apoptosis. Furthermore, UVB irradiation
alone induced predominantly apoptosis in A549 cells,
whereas combined treatment with CdSe/ZnS QDs and
UVB induced necrosis and apoptosis. The amount of LDH
released also increased significantly in a dose-dependent
manner compared to that in the control (Fig. 6(B)).
3.4. DNA Damage Indicated by the Comet and MN
Assays
We evaluated the genotoxicity induced by CdSe/ZnS QDs
with/without UVA/UVB irradiation using the comet assay,
in which the extent of DNA damage is represented by the
OTM (Fig. 7(A)). Significant DNA damage was evident
in A549 cells treated with QDs alone (OTM: 556 ± 263,
1105 ± 461, and 1510 ± 577 for control, 4.1 g/mL
(A)
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RESEARCH ARTICLE
(B)
(B)
Fig. 6. Induction of apoptosis and necrosis in A549 cells by quantum
dots (QDs) in the presence and absence of ultraviolet (UVA/UVB) light.
Apoptosis was detected using a Cell Death Detection ELISA kit (A).
Necrosis was evaluated by LDH assay (B). Values are percentages of
control, and bars represent the standard errors from duplicate experiments. Results were statistically analyzed with Dunnett’s post-hoc test
after one-way ANOVA (∗p < 005; ∗∗p < 001 compared with control).
A linear relationship between QD concentration and LDH activity was
analyzed using a linear regression model (B). The P value for a trend
was significant for LDH release (p < 005).
J. Nanosci. Nanotechnol. 12, 2160–2168, 2012
Fig. 7. DNA damage in A549 cells treated with quantum dots (QDs) in
the presence and absence of ultraviolet (UVA/UVB) light. DNA singlestrand breakage was evaluated by Olive tail moment (OTM) in a comet
assay (A) and by micronuclei formation (B). MNs, micronuclei; BNCs,
bi-nucleated cells. Values represent means ± standard error. Results were
statistically analyzed with Kruskal-Wallis and Mann-Whitney U tests.
∗
p < 005, ∗∗∗p < 0001 compared with control.
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RESEARCH ARTICLE
Cyto-/Genotoxic Effect of CdSe/ZnS Quantum Dots
Choi et al.
QDs, and 8.3 g/mL QDs, respectively), cells treated with
UVA irradiation (OTM: 807 ± 364, 1302 ± 500, and
1906 ± 633 for UVA alone, 4.1 g/mL QDs plus UVA,
and 8.3 g/mL QDs plus UVA, respectively), and cells
treated with UVB irradiation (OTM: 1235 ± 423, 1886 ±
589, and 3202 ± 866 for UVB alone, 4.1 g/mL QDs
plus UVB, and 8.3 g/mL QDs plus UVB, respectively).
UVB treatment alone induced considerable single-strand
DNA damage, and UVB irradiation significantly enhanced
the genotoxicity of CdSe/ZnS QDs.
We also performed MN assays to assess the genotoxic
effects of CdSe/ZnS QDs with/without UVA/UVB irradiation (Fig. 7(B)). The MN frequency in A549 control cells
was 31±14 MNs per 1000 bi-nucleated cells; the frequenFig. 8. Induction of reactive oxygen species in 549 cells by quantum
cies in cells treated with QDs at 1.6 g/mL and 4.1 g/mL
dots (QDs) in the presence and absence of ultraviolet (UVA/UVB) light.
were 35 ± 07 and 36 ± 21, respectively. Although QD
Bars represent mean fluorescence intensities relative to the control, and
treatment alone induced a moderate increase in MN formalines represent standard errors from triplicate experiments. Results were
tion, the frequency was remarkably increased when UVA
statistically analyzed by one-way ANOVA, followed by Dunnett’s postor UVB irradiation was combined with CdSe/ZnS QDs.
hoc test (∗p < 005).
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The MN frequencies in A549 cells with UVB irradiation
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alone (40 ± 56) and with UVB irradiation plus 1.6 g/mL
only were similar to the levels in control cells. The p53
IP : QDs
165.132.61.49
QDs (56 ± 14) and UVB irradiation plus 4.1 g/mL
mRNA
level was slightly higher in cells treated with UVB
Fri, 31 Aug 2012
02:48:17
(49 ± 28) were significantly higher than the corresponding
irradiation
alone compared with the level in control cells.
frequencies with UVA irradiation (37 ± 21, 38 ± 77, and
The
mRNA
levels of all three genes were increased in cells
42 ± 56 for UVA alone, 1.6 g/mL QDs plus UVA, and
treated with both CdSe/ZnS QDs and UVB (243 ± 11 for
4.1 g/mL QDs plus UVA, respectively).
ATM, 322 ± 13 for p53, and 48 ± 345 for caspase-9)
compared with the levels in control cells, but the differ3.5. ROS Measurement
ences were not statistically significant.
ROS induction by CdSe/ZnS QDs with and without UVA
or UVB irradiation in A549 cells was analyzed by flu4. DISCUSSION
orescence spectrophotometry. Intracellular ROS content
increased only slightly in cells treated with CdSe/ZnS
QDs with a CdSe core and ZnS shell are inorganic
QDs alone; however, significant increases in ROS formasemiconductor nanocrystals with extreme photostability,
tion were observed in cells irradiated with UVA or UVB
a broad absorption band profile, and a narrow emission
in combination with QDs. The level of ROS generation
spectrum.28 They have been widely applied for molecular
in cells treated with 1.6 g/mL QDs plus UVB irradibiomedical imaging and clinical therapies, and their potenation was significantly higher than the levels in control
tial photo-cytotoxic effects have become a topic of considcells, UVA-irradiated cells, and QD (1.6 g/mL)-treated
erable concern.29 30 In the present study, the genotoxicity
UVA-irradiated cells (Fig. 8). The results indicate that
and cytotoxicity of CdSe/ZnS QDs were comprehensively
UV irradiation, especially UVB irradiation, enhanced ROS
analyzed using several toxicological assessment methods.
generation in cells treated with CdSe/ZnS QDs.
The results showed that while CdSe/ZnS QD treatment
alone had only a mild genotoxic effect, the genotoxicity
3.6. Expression of Apoptosis Signaling Genes
and cytotoxicity of CdSe/ZnS QDs can be significantly
increased when combined with UVA or UVB irradiaA549 cells were exposed to CdSe/ZnS QDs for 4 h
tion. By assessing CdSe/ZnS QDs as a photosensitizer,
and then to UVA (2 J/cm2 or UVB (0.3 J/cm2 irradiwe demonstrated the potential effectiveness of PDT mediation. After 24 h, the mRNA levels of apoptosis-related
ated by CdSe/ZnS QDs under UVA/UVB irradiation in
genes were quantified by real-time PCR using the 2−Ct
lung cancer cells. The CdSe/ZnS QDs showed a broad
method. To investigate whether DNA damage and ROS
and continuous absorption spectrum from UV to visible
generated in cells treated with CdSe/ZnS QDs plus UVA
wavelengths (Fig. 1), whereas conventional photosensitizor UVB irradiation could induce apoptosis, the mRNA levers such as photofin, visudyne, and foscan absorb only
els for ATM, p53, and caspase-9, which are involved in the
between 630 and 700 nm.31
apoptosis signaling pathway, were analyzed (Table II). The
Two possible DNA damage induction pathways were
mRNA expression levels for ATM, p53, and caspase-9 in
cells treated with UVA irradiation only or CdSe/ZnS QDs
apparent in A549cancer cells treated with CdSe/ZnS QDs
2166
J. Nanosci. Nanotechnol. 12, 2160–2168, 2012
Choi et al.
Cyto-/Genotoxic Effect of CdSe/ZnS Quantum Dots
Table II. Quantitative analysis of ATM (A), p53 (B), and caspase-9 (C) mRNA level expression using by real-time PCR.
Concentrations (g/mL)
(-)UVA/(-)UVB
(+)UVA/(-)UVB
(-)UVA/(+)UVB
A.
Control
QD 8.3
1
1.33 ± 0.17
0.81 ± 0.19
1.16 ± 0.21
1.03 ± 0.3
2.43 ± 1.1
B.
Control
QD 8.3
1
2.26 ± 0.44
1.8 ± 0.13
2.8 ± 0.12
2.74 ± 0.25
3.22 ± 1.3
C.
Control
QD 8.3
1
1 ± 004
072 ± 002
136 ± 014
108 ± 012
48 ± 345
Note: Results were statistically analyzed with one-way ANOVA, no significant differences were found in either group (p > 0.05). Values represent the means of triplicate
determinations ± standard errors from independent experiments.
J. Nanosci. Nanotechnol. 12, 2160–2168, 2012
2167
RESEARCH ARTICLE
in the presence or absence of UVA or UVB irradiathe cell nucleus, where they interact directly with chromosomal DNA.34 35 The results from the comet and MN
tion. First, ROS generation caused by CdSe/ZnS QDs
assays support the pathway of DNA damage induction
with/without UVA/UVB elicited DNA damage. Although
through the direct interactions of CdSe/ZnS QDs with
ROS generation by CdSe/ZnS QDs was lower than that
DNA. When unrepaired single-strand breaks are converted
by classic photosensitizers,32 QDs, nevertheless,
have
charDelivered by Ingenta
to:
into double-strand breaks, micronuclei could be generated.
acteristics of a good photosensitizer, as describedYonsei
above. University
DNA double-strand breaks are known to activate ATM,
In a previous study, CdSe-core QDs, but not ZnS-coated
IP : 165.132.61.49
a serine/threonine protein kinase.36 In the present study,
CdSe-core QDs, induced apoptosis via increases
in ROS,
Fri, 31
Aug
2012
02:48:17
there was a 2.43-fold change in ATM expression in cells
cytochrome c release, and activation of caspase −9/−3.14
treated with CdSe/ZnS QDs under UVB irradiation; this
However, the current study revealed that ROS induction
could trigger p53 activation and ATM-dependent apoptoand caspase-9 mRNA expression increased considerably
sis. It is reported that the induction of apoptosis is one
in cells treated with CdSe/ZnS QDs plus UV irradiation.
of the best approaches for cancer therapy.37 Importantly,
CdSe/ZnS QD (1.6 g/mL) treatment alone induced only
although CdSe core/ZnS shell QDs are less toxic than nona mild increase in ROS (by 17%); however, ROS increases
coated CdSe core QDs, the phototoxicity of the CdSe/ZnS
were significant, up to 32% or 64%, when CdSe/ZnS
QDs was increased remarkably by UV irradiation. In this
QDs were combined with UVA or UVB, respectively. The
study, the apoptosis level was clearly higher in A549 cells
caspase-9 mRNA level also increased with UVA or UVB
treated with CdSe/ZnS QDs in combination with UVB
irradiation (1.36 or 4.8 fold-change, respectively, comirradiation (up to four times the level with CdSe/ZnS QD
pared with control). These results indicate that CdSe/ZnS
treatment alone) than in cells treated with QDs and UVA
QDs under UV irradiation act as a photosensitizer to gen(up to twice the level with CdSe/ZnS QD treatment alone).
erate ROS.
A similar trend was observed in the necrosis assay: twoROS-induced DNA damage was observed in the comet
fold increase for CdSe/ZnS QDs with UVA irradiation and
and MN assays. In the comet assay, CdSe/ZnS QD phothree-fold increase for CdSe/ZnS QDs with UVB irraditotoxicity induced by UV irradiation, especially UVB,
ation compared with QD treatment alone. The apoptosis
was statistically significant. CdSe/ZnS QD treatment with
and LDH assays could detect cytotoxicity with high sensiUVA or UVB induced single-strand DNA breaks with
tivity, whereas the MTT assay could not detect differences
a frequency that was four or six times that in the conat high QD concentrations (>8.3 g/mL CdSe/ZnS QDs).
trol, respectively, and CdSe/ZnS QDs alone induced DNA
In conclusion, our results clearly demonstrate that
single-strand breaks at a rate three times that in the consimultaneously treating mammalian cells with CdSe/ZnS
trol. Only cells treated with QDs and UVB showed a sigQDs and UV radiation elicited genotoxic and cytotoxic
nificant increase in MN formation, which is believed to
effects via DNA damage, finally inducing apoptosis and
be generated from double-strand breaks, compared with
necrosis. We elucidated a detailed picture of cytotoxicthat in the control group (Fig. 7(B)). These results suggest
ity, genotoxicity, and phototoxicity of CdSe/ZnS QD treatthat single-strand DNA damage may be repaired more effiment, UVA irradiation, and UVB irradiation, applied alone
ciently in cells treated with CdSe/ZnS QDs and UVA irraor in combination. The results suggest the potential effidiation than in UVB-irradiated cells. Because UVB light
cacy and general guidelines for using CdSe/ZnS QDs and
has more mutagenic effect than UVA light, DNA damage
UV irradiation during PDT for cancer.
caused by UVB is difficult to repair, thereby causing more
damage.33
Acknowledgments: This research was supported by
The second possible DNA damage induction pathway
Basic Science Research Program [2010-0013018;2011involves the entry of endocytosed CdSe/ZnS QDs into
0004942] through the National Research Foundation of
Cyto-/Genotoxic Effect of CdSe/ZnS Quantum Dots
Choi et al.
RESEARCH ARTICLE
Korea (NRF); Brain Korea 21program funded by the
Ministry of Education, Science and Technology.
15. A. O. Choi, S. J. Cho, J. Desbarats, J. Lovric, and D. Maysinger,
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5. CONFLICTS OF INTERESTS
18. A. Chiaviello, I. Paciello, I. Postiglione, E. Crescenzi, and
G. Palumbo, Cell prolif. 43, 480 (2010).
The authors report no conflict of interest. We are respon19. L. Collins, C. Haines, R. Perkel, and R. Enck, Am. Fam. Physician
sible for the content and writing of this paper.
75, 56 (2007).
20. P. A. Jr. Bunn and N. Thatcher, Oncologist 13, 37 (2008).
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Received: 17 November 2011. Accepted: 18 November 2011.
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