Cyto-/Genotoxic Effect of CdSe/ZnS Quantum Dots in …web.yonsei.ac.kr/bionano/Publication/41. (J Nanosci Nanotech,2012... · advantages accompany nano-scale biomaterials, which typ-

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Copyright © 2012 American Scientific PublishersAll rights reservedPrinted in the United States of America

Journal ofNanoscience and Nanotechnology

Vol. 12, 2160–2168, 2012

Cyto-/Genotoxic Effect of CdSe/ZnS Quantum Dots inHuman 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�∗

1School of Public Health and Institute of Health and Environment, Seoul National University, 1 Gwanak-ro,Gwanak-gu, Seoul, 151-742, Republic of Korea

2Chung-Ang University, Heukseok-dong, Dongjak-gu, Seoul, 156-756, Republic of Korea3Translational Research Center for Protein Function Control & Department of Materials Science and Engineering,

Yonsei University, Seoul, 120-749, Republic of Korea

Quantum dots (QDs) are luminescent nanoparticles (NPs) with promising potential in numerousmedical applications, but there remain persistent human health and safety concerns. Althoughthe cytotoxic effects of QDs have been extensively investigated, their genotoxic effects remainunder-explored. This study scrutinized the cyto- and genotoxic effects of QDs with a Cadmiumselenide/Zinc sulfide (CdSe/ZnS) core/shell, and suggests comprehensive guidelines for the appli-cation of QDs in cancer therapy. QDs were used to treat A549 cells in the presence andabsence of ultraviolet A/B (UVA/UVB) irradiation. QD-induced cell death was evaluated by 3-(4,5-Dimethylthiazol-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, suchas ataxia telangiectasia mutated (ATM), p53, and caspase-9, involved in apoptosis. The genotoxiceffect of CdSe/ZnS QDs was measured in human cancer cells, for the first time, by comet andmicronucleus assays. Treatment with CdSe/ZnS QDs and UVB irradiation resulted in the mostsevere extent of cell death, indicating strong induction of phototoxicity by CdSe/ZnS QDs in thepresence of UVB. Both apoptotic and necrotic cell death were observed upon QDs and UVB com-bined treatment. The induction of Olive tail moments and micronuclei formation was also mostsignificant when CdSe/ZnS QDs and UVB irradiation were combined. Our results on the genotoxiceffect and mechanistic details of CdSe/ZnS QD-induced cell death suggest that UVB irradiation isthe 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 promisingand attractive fields of nanotechnology.1–3 Nanometer-sized materials have unique and valuable properties, com-pared with conventional bulk materials, and numerousadvantages accompany nano-scale biomaterials, which typ-ically range in size from tens to hundreds of nanome-ters. Nanotechnology-based photodynamic therapy (PDT),one of the most promising cancer-treatment nanotech-nologies, is noninvasive and fairly safe for repeated

∗Author to whom correspondence should be addressed.

administration.4 Moreover, a tumor-specific targeting strat-egy could be developed with appropriate functionalizationof nanoparticles (NPs).PDT uses special drugs, called photosensitizing agents,

and visible or near-infrared illumination at specific wave-lengths to kill cancer cells. Reactive oxygen intermediatesare generated during photoactivation of a photosensitizer(PS) drug, and these immediately react with and dam-age vital biomolecules in cell organelles, causing celldeath.5 PDT was first used to treat cancer more than100 years ago; however, in the earlier days, the adminis-tration of porphyrins and phthalocyanines induced severeside effects attributable to non-specific drug delivery andphotoactivation.6

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Choi et al. Cyto-/Genotoxic Effect of CdSe/ZnS Quantum Dots

Quantum dots (QDs), which are inorganic semiconduc-tor nanocrystals, have become one of the most attrac-tive nanomaterials for bioimaging and cancer therapy.The development of QD technology has paved the wayfor using QDs as agents in medical diagnostics, cancerdiagnosis, and PDT for cancer. QD technology has beenapplied in many types of animal models such as mouseand pig models.7�8 The most salient material propertiesof QDs include their bright fluorescence emission, excep-tional photostability, large surface area, and large two-photon absorption cross-section.9–12

Previous studies have shown that QDs with a Cadmiumselenide (CdSe) core induce cell death by increasingreactive oxygen species (ROS) and inhibiting survival-related signaling events.13 QDs also induce apoptosis viamitochondrial-dependent pathways involving Fas upreg-ulation and lipid peroxidation in human neuroblastomacells.14�15 The cytotoxicity of CdSe-core QDs is correlatedwith the release of free Cd2+. One of the most efficientways of reducing QD toxicity is to cover the CdSe corewith a Zinc sulfide (ZnS) coating layer. In the presentstudy, the ZnS shell plays an important role as a barrierto prevent CdSe from contacting the surrounding solvent,thus protecting the CdSe core against dissolution throughionization. The ZnS shell also increases the QD quantumyield.14

Despite the widespread use of QDs in diverse biomed-ical fields, including imaging, diagnosis, and PDT, adetailed picture of the cellular effects of Cadmiumselenide/Zinc sulfide (CdSe/ZnS) QDs has not been estab-lished. Here, we investigated the cyto-/genotoxic effectsand mechanistic details of the cell death pathway inducedin human lung adenocarcinoma cells (A549 cells) byCdSe/ZnS QDs in the presence and absence of UV irra-diation. A549 cells are commonly used in cancer studies,including PDT studies.16–18 Lung cancer is the lead-ing cause of cancer deaths, with a cure rate of only13% and patients do not survive 2 years owing to latedetection.19�20 It is reported that engineered nanoparticles,such as silver nanoparticles (Ag NP), nano titanium diox-ide (TiO2), and carbon nanotube (CNT), can cause lungdamage via enhanced inflammatory responses, lung func-tion change, and fibrosis of lung tissue, finally inducinglung cancer.21–23

To determine the conditions in which QDs can be mosteffective as a PDT photosensitizer, we tested the cytotox-icity of CdSe core/ZnS shell QDs with and without UVirradiation in A549 cells using MTT, lactate dehydroge-nase (LDH), and apoptosis assays.The genotoxicity induced by CdSe/ZnS QDs in human

lung cancer cells was measured primarily by micronucleusand comet assays. Furthermore, we analyzed differentialmRNA levels of genes involved in apoptosis, to investigatethe global effect of CdSe/ZnS QDs and UV irradiation onA549 cells.

2. MATERIAL AND METHODS

2.1. Cell Culture

Adenocarcinoma human alveolar basal epithelial cells(A549 cells) were obtained from the Korean Cell LineBank (Seoul, Korea) and cultured in RPMI 1640 supple-mented with filtered 10% fetal bovine serum, penicillin,and streptomycin (100 U/mL of each), at 37 �C in ahumidified atmosphere of 5% CO2. CdSe/ZnS quantumdot (Lumidot™) was purchased from Sigma-Aldrich.

2.2. Preparation and Characterization ofCdSe/ZnS QDs

CdSe/ZnS QDs dispersed in toluene were supplied bySigma-Aldrich (St. Louis, MO, USA). Toluene wasremoved via rotary evaporation (Rotavapor R-210; BuchiLaboratory Equipment, Zurich, Switzerland), and the QDswere suspended in distilled water. This solution was soni-cated for 30 min at 4 �C before each experiment. Dynamiclight scattering (DLS) is an indirect method for measur-ing particle size. Briefly, the fluctuations in scattered lightintensity, which are caused by Brownian motion of sus-pended particles, are measured over time. DLS was mea-sured at room temperature with an ALV/CGS-3 CompactGoninometer System (Hessen, Germany) equipped with anHe-Ne laser operating at 632.8 nm. The scattering anglewas 90�. The size distribution was determined using aconstrained regularization method. Transmission electronmicroscopy (TEM; 100CX; JEOL, Tokyo, Japan) was usedto determine the size and shape of the CdSe/ZnS QDs.Absorption peaks of the CdSe/ZnS QDs were measuredusing an ultraviolet-visible (UV-Vis) spectrophotometer(V-650; Jasco, Tokyo, Japan), and photoluminescencespectra were detected with a fluorescence spectropho-tometer (F-4500; Hitachi, Tokyo, Japan). The QD sur-face charge was measured in distilled water using azeta potential analyzer (Zeta plus; Brookhaven Instru-ments Corp., Holtsville, NY, USA). Scanning electronmicroscopy (SEM), with an energy dispersive X-ray(EDX) spectrometer as a detector (AXS; Bruker, Bad Her-renalb, Germany), was used to identify the major ele-ment types in A549 cells. Cells were counterstained withDAPI (4, 6-diamidino-2-phenylindole)-containing mount-ing medium to visualize cellular uptake of CdSe/ZnSQDs by fluorescence microscopy (Vectorshield, Peterbor-ough, UK). Confocal luminescence images of A549 cellswere obtained using a confocal microscope (Carl Zeiss,Oberkochen, Germany) equipped with a 405-nm diodelaser and 488-nm Ar laser.

2.3. QD Cytotoxicity

The cytotoxic effects induced by the CdSe/ZnS QDsin A549 cells were evaluated by MTT (3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyl-tetrazolium bromide)

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assay. A549 cells were seeded in a 96-well plate at a den-sity of 10,000 cells per well and incubated for 24 h at37 �C in a humidified atmosphere of 5% CO2. Then, thecells were treated with CdSe/ZnS QDs at different con-centrations with/without UVA or UVB irradiation. Aftera 24-h incubation, the cells were treated with 0.5 mg/mLMTT solution (formazan powder; Sigma Aldrich), andthe plate was incubated for 4 h. The violet formazancrystals were dissolved in DMSO and the absorbance at560 nm was measured using a microplate reader (Tecan,Männedorf, Switzerland).

2.4. UVA/UVB Irradiation

Cells were plated in growth medium in a 96-well plateand exposed to UVA (2 J/cm2� or UVB (0.3 J/cm2� light,using a Bio-Link BLX irradiation system (Vilber-Lourmat,Marne-la-Vallee Cdex 1, France) equipped with five 8 WUV lamps (UV-A Vilber-Lourmat T-8L with peak irradi-ance at 365; UV-B Vilber-Lourmat T-8M with peak irra-diance at 312 nm). Cell viability was determined by MTTassay.

2.5. Single Cell Gel Electrophoresis Assay

Single-cell gel electrophoresis was performed as describedpreviously by Singh et al.24 A549 cells were treated withCdSe/ZnS QDs for 3 h, followed by UVA (2 J/cm2) orUVB (0.3 J/cm2) exposure. After being washed twice withphosphate-buffered saline (PBS), the cells were maintainedat 4 �C to prevent DNA repair. Slides were preparedaccording to the method of Singh.24 Briefly, images of 60cells randomly selected from each sample were analyzed,using a Komet 5.5 image analysis system (Kinetic ImagingLtd., Nottingham, UK). The Olive tail moment (OTM) ofeach cell was measured under a fluorescence microscope(Nikon, Tokyo, Japan) equipped with a 515- to 560 nmexcitation filter and 590 nm barrier filter.

2.6. Cytokinesis-Block Micronucleus Assay

The cytokinesis-block micronucleus (MN) assay was per-formed as described by Fenech.25 A549 cells were cul-tured in RPMI 1640 medium supplemented with 10%fetal bovine serum, incubated with CdSe/ZnS QDs for3 h, and then irradiated with UVA or UVB. Cytocha-lasin B (4 �g/mL; Sigma-Aldrich) was added 20 h afterthe start of culture, and the incubation was continuedfor an additional 28 h. After a total incubation periodof 48 h, the cells were harvested, treated with hypotonic0.075 M KCl solution for 1 min, and washed twice withfixative solution (methanol:acetic acid, 3:1). Air-dried cellpreparations were stained with Giemsa solution (5%). Intotal, 1,000 binucleated cells with well-preserved cyto-plasm were scored according to standard criteria.26

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; MolecularProbes, Eugene, OR, USA). A549 cells were treated withQDs for 2 h, and then they were irradiated with UVAor UVB. After 1 h, the samples were centrifuged andadded with new medium containing 20 �M DCFDA inthe dark. The cells were incubated for 20 min at 37 �Cand monitored using a fluorescence spectrophotometer at535 nm emission with an excitation wavelength of 485 nm(Tecan, Männedorf, Switzerland).27

2.8. Measurement of Apoptosis and Necrosis

Apoptosis induced by CdSe/ZnS QDs with or withoutUVA or UVB irradiation was assessed using a termi-nal deoxynucleotidyl transferase dUTP nick end label-ing apoptosis assay (Cell Death Detection ELISAPLUS kit;Roche Applied Science, Mannheim, Germany), accord-ing to the manufacturers’ protocol. Briefly, A549 cellswere treated with CdSe/ZnS QDs, followed by UVA orUVB irradiation. After 24 h, the culture supernatant wasremoved, and the cells were incubated with lysis buffer.After centrifugation, an aliquot of the supernatant wastransferred to a streptavidin-coated microplate well andincubated with anti-histone and anti-DNA antibodies for2 h at room temperature, to bind nucleosomes in the super-natant. The immobilized immunocomplexes were washedthree times to remove cell components and were detectedby incubation with peroxidase substrate. Absorbance at405 nm was measured using a microplate reader.Necrosis was determined by measuring LDH released

into the medium by necrotic cells upon plasma membranerupture (Cytotoxicity Detection KitPLUS, Roche AppliedScience). Cells were cultured as for the apoptosis assay.Briefly, substrate reagent was incubated with the culturesupernatant, and the absorbance at 490 nm was read usinga microplate reader.

2.9. RNA Extraction and Quantitative RealTime-Polymerase Chain Reaction (PCR)

Total RNA was extracted from A549 cells treated withCdSe/ZnS QDs in the presence or absence of UVA or UVBirradiation, using an AxyPrep Multisource Total RNAMiniprep kit (Axygen Biosciences, Union City, CA, USA)according to the manufacturer’s instructions. For cDNAsynthesis, total RNA was reverse transcribed using a ran-dom primer and a reverse transcription system (Promega,Madison, WI, USA). Real-time PCR was performed usingan Applied Biosystems 7300 real-time PCR system (FosterCity, CA, USA) and Express Start SYBR Green qPCRSupermix (Invitrogen, Burlington, ON, Canada). The syn-thesized primer sequences for the reactions are shown

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Table I. Primer sequences used for quantitative real-time PCR.

Primer pair Sequence (5′-3′)

ATM -CGAGGTGAGCGGATCACAA-; -TTGGCCCACAGCAACCTT-p53 -GTGAGCGCTTCGAGATGTTC-; -ATGGCGGGAGGTAGACTGAC-caspase-9 -GCTCTTCCTTTGTTCATCTCC-; -CATCTGGCTCGGGGTTACTGC–GAPDH (endogenous control) -GGAAGGTGAAGGTCGGAGTCA-; -GTCATTGATGGCAACAATATCCACT-

in Table I. The thermal cycling conditions were incubationat 94 �C for 5 min, followed by 40 cycles of 94 �C for15 s, 55 �C for 30 s, and 72 �C for 30 s. Each assay wasperformed in triplicate, with GAPDH as an endogenouscontrol for normalizing expression levels.Data are expressed as the ratios of ataxia telangiectasia

mutated (ATM), p53, and caspase-9 to GAPDH, accordingto the comparative threshold cycle method (2−��Ct).

2.10. Statistical Analysis

The effects of CdSe/ZnS QDs with or without UVAor UVB irradiation on DNA were analyzed using non-parametric Kruskal–Wallis one-way analysis of vari-ance (ANOVA) and the Mann–Whitney U test. One-wayANOVA followed by Dunnett’s or Tukey’s test was appliedto analyze cell viability, ROS generation, induction ofapoptosis and necrosis, and mRNA expression, using SPSSsoftware v. 12 (SPSS Inc., Chicago, IL, USA). Lineartrends for the induction of apoptosis and necrosis weretested using a linear regression model.

3. RESULTS

3.1. Physicochemical Characterization andIntracellular 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 solu-tion. Toluene was removed by rotary evaporator to com-plete dryness. Then the QDs were suspended in distilledwater, and the physicochemical and photophysical proper-ties of aqueous QDs were analyzed by DLS, UV-Vis spec-troscopy, steady-state fluorescence emission spectroscopy,zeta potential, and TEM. The average size of CdSe/ZnSQDs was 105±15�55 nm as determined by DLS, indicat-ing that QD particles are aggregated together in aqueousenvironment. However, the size of QD aggregates is stillin the nano-regime, which is small enough for efficientendocytic entry into cells. The UV-Vis absorption spec-trum of CdSe/ZnS QDs showed broad continuous absorp-tion at wavelengths extending from the UV to the visiblerange (Fig. 1(A)). The photoluminescence emission spec-trum was narrow, with maximum emission at 535 nm(Fig. 1(B)). These results indicate that photophysical prop-erties of the QDs are largely preserved in aqueous envi-ronment. The zeta potential measurement in distilled water

showed a � potential of +40�58±7�97 mV, confirming thecolloidal stability of CdSe/ZnS QDs. The shape and sizeof the QDs were also analyzed by TEM, which revealedthat the diameter of individual CdSe/ZnS QD nanopar-ticles in the aggregates is approximately 4 nm (Fig. 2).The CdSe/ZnS QD particles internalized after a 4-h celltreatment were examined by EDX spectroscopy (Fig. 3).Spectral peaks demonstrated the presence of Cd, Se, Zn,and S. Cellular uptake was further confirmed by confocalmicroscopy, which also showed the intracellular localiza-tion of CdSe/ZnS QDs in A549 cells. Cells were treatedwith 8.3 �g/mL QDs, and confocal Z-stack analysis ofthe treated cells verified the intracellular translocation ofCdSe/ZnS QDs (Fig. 4).

(A)

(B)

Fig. 1. Characterization of CdSe/ZnS quantum dots (QDs). UV-Visabsorption spectrum of QDs (A). Photoluminescence (PL) spectrum(emission wavelength = 535 nm) of QDs. The maximum emissionoccurred at a wavelength of 535 nm (B).



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Fig. 2. Image of transmission electron microscope of QDs. Scale bar is50 nm. Magnification: × 400,000. The inset shows a higher resolution.Agglomerates of QDs are observed.

3.2. Phototoxicity of QDs in A549 Cells under UVIrradiation

MTT assays were performed in A549 cells to assessCdSe/ZnS QD cytotoxicity with and without UVA or UVBirradiation. An overall dose-dependent decrease in cell via-bility was observed. The effect of CdSe/ZnS QD pho-toactivation on cell viability was more significant whenthe cells were irradiated with UVB than with UVA irra-diation (Fig. 5). The A549 cells were more suscepti-ble to the effect of CdSe/ZnS QDs combined with UVBirradiation (68.81% cell viability) than to the effects ofCdSe/ZnS QDs plus UVA irradiation (82.01% cell via-bility) or CdSe/ZnS QDs (1.6 �g/mL) alone (89.2% cellviability).

Fig. 3. Energy dispersive X-ray (EDX) specta of CdSe/ZnS quantumdots. Confirming the presence of Cd, Se, Zn, and S. Measuring time:5 min. High voltage: 10 keV.

Fig. 4. Confocal images of internalized quantum dots (QDs) inA549 cells. Magnification: ×630. 3-D Z-stack image shows the localiza-tion of the QDs in A549 cells. Green, QDs; blue, cell nuclei stained withDAPI. Arrows indicate the QDs.

3.3. Assessment of Apoptosis and Necrosis

We next conducted apoptosis assays to investigate themode of CdSe/ZnS QD-induced cell death in the presenceor absence of UVA or UVB irradiation. We used a one-stepsandwich ELISA assay in which mouse monoclonal anti-bodies directed against DNA and histones immunoreactwith mono- and oligonucleosomes in the cytoplasmic frac-tions of cell lysates to colorimetrically quantify the relativelevel of apoptosis. This assay showed that CdSe/ZnS QDs

Fig. 5. Cell viability assay for quantum dots (QDs) in the presenceand absence of ultraviolet (UV) light. A549 cells were incubated in a96-well microplate for 24 h and then treated with QDs with and with-out UVA/UVB. Cell viability was determined by MTT assay. Data arepercentages of control, and error bars represent standard errors fromtriplicate experiments. Results were statistically analyzed with Dunnett’spost-hoc test (∗∗p < 0�01 compared with control). Letters denote signifi-cant differences among treatments using Tukey’s HSD test after one-wayANOVA (p < 0�05).



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induced A549 cell apoptosis in a dose-dependent manner(Fig. 6(A)), with a more significant increase in apoptosisgenerally observed in cells treated with both CdSe/ZnSQDs and UVB. Interestingly, the induction of apoptosisin the UVB-treated group was somewhat reduced at thehighest QD concentration (16.6 �g/mL).In contrast, the LDH assay, which is a sensitive detec-

tion method for necrotic cell death, revealed significantand consistent increases in LDH release in proportionto increases in QD concentration (Fig. 6(B)). Takentogether, the results indicate that CdSe/ZnS QDs at rela-tively low concentrations induced apoptosis, but QDs at

(A)

(B)

Fig. 6. Induction of apoptosis and necrosis in A549 cells by quantumdots (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 ofcontrol, and bars represent the standard errors from duplicate experi-ments. Results were statistically analyzed with Dunnett’s post-hoc testafter one-way ANOVA (∗p < 0�05; ∗∗p < 0�01 compared with control).A linear relationship between QD concentration and LDH activity wasanalyzed using a linear regression model (B). The P value for a trendwas significant for LDH release (p < 0�05).

higher concentrations had a tendency to induce necro-sis, rather than apoptosis. Furthermore, UVB irradiationalone induced predominantly apoptosis in A549 cells,whereas combined treatment with CdSe/ZnS QDs andUVB induced necrosis and apoptosis. The amount of LDHreleased also increased significantly in a dose-dependentmanner compared to that in the control (Fig. 6(B)).

3.4. DNA Damage Indicated by the Comet and MNAssays

We evaluated the genotoxicity induced by CdSe/ZnS QDswith/without UVA/UVB irradiation using the comet assay,in which the extent of DNA damage is represented by theOTM (Fig. 7(A)). Significant DNA damage was evidentin A549 cells treated with QDs alone (OTM: 5�56±2�63,11�05± 4�61, and 15�10± 5�77 for control, 4.1 �g/mL

(A)

(B)

Fig. 7. DNA damage in A549 cells treated with quantum dots (QDs) inthe presence and absence of ultraviolet (UVA/UVB) light. DNA single-strand breakage was evaluated by Olive tail moment (OTM) in a cometassay (A) and by micronuclei formation (B). MNs, micronuclei; BNCs,bi-nucleated cells. Values represent means± standard error. Results werestatistically analyzed with Kruskal-Wallis and Mann-Whitney U tests.∗p < 0�05, ∗∗∗p < 0�001 compared with control.



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QDs, and 8.3 �g/mL QDs, respectively), cells treated withUVA irradiation (OTM: 8�07± 3�64, 13�02± 5�00, and19�06± 6�33 for UVA alone, 4.1 �g/mL QDs plus UVA,and 8.3 �g/mL QDs plus UVA, respectively), and cellstreated with UVB irradiation (OTM: 12�35±4�23, 18�86±5�89, and 32�02± 8�66 for UVB alone, 4.1 �g/mL QDsplus UVB, and 8.3 �g/mL QDs plus UVB, respectively).UVB treatment alone induced considerable single-strandDNA damage, and UVB irradiation significantly enhancedthe genotoxicity of CdSe/ZnS QDs.We also performed MN assays to assess the genotoxic

effects of CdSe/ZnS QDs with/without UVA/UVB irradia-tion (Fig. 7(B)). The MN frequency in A549 control cellswas 31±1�4 MNs per 1000 bi-nucleated cells; the frequen-cies in cells treated with QDs at 1.6 �g/mL and 4.1 �g/mLwere 35± 0�7 and 36± 2�1, respectively. Although QDtreatment alone induced a moderate increase in MN forma-tion, the frequency was remarkably increased when UVAor UVB irradiation was combined with CdSe/ZnS QDs.The MN frequencies in A549 cells with UVB irradiationalone (40±5�6) and with UVB irradiation plus 1.6 �g/mLQDs (56±1�4) and UVB irradiation plus 4.1 �g/mL QDs(49±2�8) were significantly higher than the correspondingfrequencies with UVA irradiation (37±2�1, 38±7�7, and42± 5�6 for UVA alone, 1.6 �g/mL QDs plus UVA, and4.1 �g/mL QDs plus UVA, respectively).

3.5. ROS Measurement

ROS induction by CdSe/ZnS QDs with and without UVAor UVB irradiation in A549 cells was analyzed by flu-orescence spectrophotometry. Intracellular ROS contentincreased only slightly in cells treated with CdSe/ZnSQDs alone; however, significant increases in ROS forma-tion were observed in cells irradiated with UVA or UVBin combination with QDs. The level of ROS generationin cells treated with 1.6 �g/mL QDs plus UVB irradi-ation was significantly higher than the levels in controlcells, UVA-irradiated cells, and QD (1.6 �g/mL)-treatedUVA-irradiated cells (Fig. 8). The results indicate thatUV irradiation, especially UVB irradiation, enhanced ROSgeneration in cells treated with CdSe/ZnS QDs.

3.6. Expression of Apoptosis Signaling Genes

A549 cells were exposed to CdSe/ZnS QDs for 4 hand then to UVA (2 J/cm2� or UVB (0.3 J/cm2� irradi-ation. After 24 h, the mRNA levels of apoptosis-relatedgenes were quantified by real-time PCR using the 2−��Ct

method. To investigate whether DNA damage and ROSgenerated in cells treated with CdSe/ZnS QDs plus UVAor UVB irradiation could induce apoptosis, the mRNA lev-els for ATM, p53, and caspase-9, which are involved in theapoptosis signaling pathway, were analyzed (Table II). ThemRNA expression levels for ATM, p53, and caspase-9 incells treated with UVA irradiation only or CdSe/ZnS QDs

Fig. 8. Induction of reactive oxygen species in 549 cells by quantumdots (QDs) in the presence and absence of ultraviolet (UVA/UVB) light.Bars represent mean fluorescence intensities relative to the control, andlines represent standard errors from triplicate experiments. Results werestatistically analyzed by one-way ANOVA, followed by Dunnett’s post-hoc test (∗p < 0�05).

only were similar to the levels in control cells. The p53mRNA level was slightly higher in cells treated with UVBirradiation alone compared with the level in control cells.The mRNA levels of all three genes were increased in cellstreated with both CdSe/ZnS QDs and UVB (2�43±1�1 forATM, 3�22± 1�3 for p53, and 4�8± 3�45 for caspase-9)compared with the levels in control cells, but the differ-ences were not statistically significant.

4. DISCUSSION

QDs with a CdSe core and ZnS shell are inorganicsemiconductor nanocrystals with extreme photostability,a broad absorption band profile, and a narrow emissionspectrum.28 They have been widely applied for molecularbiomedical imaging and clinical therapies, and their poten-tial photo-cytotoxic effects have become a topic of consid-erable concern.29�30 In the present study, the genotoxicityand cytotoxicity of CdSe/ZnS QDs were comprehensivelyanalyzed using several toxicological assessment methods.The results showed that while CdSe/ZnS QD treatmentalone had only a mild genotoxic effect, the genotoxicityand cytotoxicity of CdSe/ZnS QDs can be significantlyincreased when combined with UVA or UVB irradia-tion. By assessing CdSe/ZnS QDs as a photosensitizer,we demonstrated the potential effectiveness of PDT medi-ated by CdSe/ZnS QDs under UVA/UVB irradiation inlung cancer cells. The CdSe/ZnS QDs showed a broadand continuous absorption spectrum from UV to visiblewavelengths (Fig. 1), whereas conventional photosensitiz-ers such as photofin, visudyne, and foscan absorb onlybetween 630 and 700 nm.31

Two possible DNA damage induction pathways wereapparent in A549cancer cells treated with CdSe/ZnS QDs



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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 1 0.81 ± 0.19 1.03 ± 0.3QD 8.3 1.33 ± 0.17 1.16 ± 0.21 2.43 ± 1.1

B.Control 1 1.8 ± 0.13 2.74 ± 0.25QD 8.3 2.26 ± 0.44 2.8 ± 0.12 3.22 ± 1.3

C.Control 1 0�72±0�02 1�08±0�12QD 8.3 1±0�04 1�36±0�14 4�8±3�45

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 triplicatedeterminations ± standard errors from independent experiments.

in the presence or absence of UVA or UVB irradia-tion. First, ROS generation caused by CdSe/ZnS QDswith/without UVA/UVB elicited DNA damage. AlthoughROS generation by CdSe/ZnS QDs was lower than thatby classic photosensitizers,32 QDs, nevertheless, have char-acteristics of a good photosensitizer, as described above.In a previous study, CdSe-core QDs, but not ZnS-coatedCdSe-core QDs, induced apoptosis via increases in ROS,cytochrome c release, and activation of caspase −9/−3.14

However, the current study revealed that ROS inductionand caspase-9 mRNA expression increased considerablyin cells treated with CdSe/ZnS QDs plus UV irradiation.CdSe/ZnS QD (1.6 �g/mL) treatment alone induced onlya mild increase in ROS (by 17%); however, ROS increaseswere significant, up to 32% or 64%, when CdSe/ZnSQDs were combined with UVA or UVB, respectively. Thecaspase-9 mRNA level also increased with UVA or UVBirradiation (1.36 or 4.8 fold-change, respectively, com-pared with control). These results indicate that CdSe/ZnSQDs under UV irradiation act as a photosensitizer to gen-erate ROS.ROS-induced DNA damage was observed in the comet

and MN assays. In the comet assay, CdSe/ZnS QD pho-totoxicity induced by UV irradiation, especially UVB,was statistically significant. CdSe/ZnS QD treatment withUVA or UVB induced single-strand DNA breaks witha frequency that was four or six times that in the con-trol, respectively, and CdSe/ZnS QDs alone induced DNAsingle-strand breaks at a rate three times that in the con-trol. Only cells treated with QDs and UVB showed a sig-nificant increase in MN formation, which is believed tobe generated from double-strand breaks, compared withthat in the control group (Fig. 7(B)). These results suggestthat single-strand DNA damage may be repaired more effi-ciently in cells treated with CdSe/ZnS QDs and UVA irra-diation than in UVB-irradiated cells. Because UVB lighthas more mutagenic effect than UVA light, DNA damagecaused by UVB is difficult to repair, thereby causing moredamage.33

The second possible DNA damage induction pathwayinvolves the entry of endocytosed CdSe/ZnS QDs into

the cell nucleus, where they interact directly with chro-mosomal DNA.34�35 The results from the comet and MNassays support the pathway of DNA damage inductionthrough the direct interactions of CdSe/ZnS QDs withDNA. When unrepaired single-strand breaks are convertedinto double-strand breaks, micronuclei could be generated.DNA double-strand breaks are known to activate ATM,a serine/threonine protein kinase.36 In the present study,there was a 2.43-fold change in ATM expression in cellstreated with CdSe/ZnS QDs under UVB irradiation; thiscould trigger p53 activation and ATM-dependent apopto-sis. It is reported that the induction of apoptosis is oneof the best approaches for cancer therapy.37 Importantly,although CdSe core/ZnS shell QDs are less toxic than non-coated CdSe core QDs, the phototoxicity of the CdSe/ZnSQDs was increased remarkably by UV irradiation. In thisstudy, the apoptosis level was clearly higher in A549 cellstreated with CdSe/ZnS QDs in combination with UVBirradiation (up to four times the level with CdSe/ZnS QDtreatment alone) than in cells treated with QDs and UVA(up to twice the level with CdSe/ZnS QD treatment alone).A similar trend was observed in the necrosis assay: two-fold increase for CdSe/ZnS QDs with UVA irradiation andthree-fold increase for CdSe/ZnS QDs with UVB irradi-ation compared with QD treatment alone. The apoptosisand LDH assays could detect cytotoxicity with high sensi-tivity, whereas the MTT assay could not detect differencesat high QD concentrations (>8.3 �g/mL CdSe/ZnS QDs).In conclusion, our results clearly demonstrate that

simultaneously treating mammalian cells with CdSe/ZnSQDs and UV radiation elicited genotoxic and cytotoxiceffects via DNA damage, finally inducing apoptosis andnecrosis. We elucidated a detailed picture of cytotoxic-ity, genotoxicity, and phototoxicity of CdSe/ZnS QD treat-ment, UVA irradiation, and UVB irradiation, applied aloneor in combination. The results suggest the potential effi-cacy and general guidelines for using CdSe/ZnS QDs andUV irradiation during PDT for cancer.

Acknowledgments: This research was supported byBasic Science Research Program [2010-0013018;2011-0004942] through the National Research Foundation of



Fri, 31 Aug 2012 02:48:17

RESEARCH

ARTIC

LE


Korea (NRF); Brain Korea 21program funded by theMinistry of Education, Science and Technology.

5. CONFLICTS OF INTERESTS

The authors report no conflict of interest. We are respon-sible for the content and writing of this paper.

References and Notes

1. M. Pautler and S. Brenner, Int. J. Nanomedicine. 5, 803 (2010).2. C. Fang and M. Zhang, J. Control. Release. 146, 2 (2010).3. E. Blanco, A. Hsiano, A. P. Mann, M. G. Landry, F. Meric-Bernstam,

and M. Ferrari, Cancer. Sci. 102, 1247 (2011).4. R. Misra, S. Acharya, and S. K. Sahoo, Drug. Discov. Today 15, 842

(2010).5. V. Biju, S. Mudayoor, R. V. Omkumar, A. Anas, and M. Ishikawa,

Biotechnol. Adv. 28, 199 (2010).6. U. O. Nseyo, J. Dehaven, T. J. Dougherty, W. R. Potter, D.L. Merrill,

S. L. Lundahl, and D. L. Lamm, J. Clin. Laser. Med. Surg. 16, 61(1998).

7. W. Jiang, E. Papa, H. Fischer, S. Mardyani, and W. C. W. Chan,Trends. Biotechnol. 22, 607 (2004).

8. A. M. Iga, J. H. P. Robertson, C. Winslet, and A. M. Seifalian,J. Biomed. Biotechnol. 2007, 76087 (2007).

9. A. C. S. Samia, X. B. Chen, and C. Burda, J. Am. Chem. Soc. 125,15736 (2003).

10. J. Lovric, S. J. Cho, F. M. Winnik, and D. Maysinger, Chem. Biol.12, 1227 (2005).

11. A. Anas, H. Akita, H. Harashima, T. Itoh, M. Ishikawa, and V. Biju,J. Phys. Chem. B. 112, 10005 (2008).

12. E. Yaghini, A. M. Seifalian, and A. J. MacRobert, Nanomedicine 4,353 (2009).

13. M. S. Hsieh, N. H. Shiao, and W. H. Chan, Int. J. Mol. Sci. 10, 2122(2009).

14. W. H. Chan, N. H. Shiao, and P. Z. Lu, Toxicol. Lett. 167, 191(2006).

15. A. O. Choi, S. J. Cho, J. Desbarats, J. Lovric, and D. Maysinger,J. Mol. Med. 86, 291 (2007).

16. M. Korbelik and G. Krosl, Photochem. Photobiol. 60, 497 (1994).17. P. Sharma, T. Farrell, M. S. Patterson, G. Singh, J. R. Wright, R. Sur,

and A. J. Rainbow, Photochem. Photobiol. 85, 99 (2009).18. A. Chiaviello, I. Paciello, I. Postiglione, E. Crescenzi, and

G. Palumbo, Cell prolif. 43, 480 (2010).19. L. Collins, C. Haines, R. Perkel, and R. Enck, Am. Fam. Physician

75, 56 (2007).20. P. A. Jr. Bunn and N. Thatcher, Oncologist 13, 37 (2008).21. J. H. Sung, J. H. Ji, J. U. Yoon, D. S. Kim, J. H. Lee, and I. J. Yu,

Inhal. Toxicol. 20, 567 (2008).22. D. B. Warheit, Anal. Bioanal. Chem. 398, 607 (2010).23. J. C. Bonner, Proc. Am. Thorac. Soc. 7, 138 (2010).24. N. P. Singh, M. T. McCoy, R. R. Tice, and E. L. Schneider, Exp.

Cell. Res. 175, 184 (1988).25. M. Fenech, Mutat. Res. 285, 35 (1993).26. M. Fenech, Mutat. Res. 455, 81 (2000).27. M . Tepel, Kidney International 58, 867 (2000).28. A. R. Santos, A. S. Miguel, L. Tomaz, R. Malho, C. Maycock,

M. C. V. Patto, P. Fevereiro, and A. Oliva, J. Nanobiotechnology 8,24 (2010).

29. W. K. B. Khalil, E. Girgis, A. N. Emam, M. B. Mohamed, and K. V.Rao, Chem. Res. Toxicol. 24, 640 (2011).

30. J. Kim, Y. Park, T. H. Yoon, C. S. Yoon, and K. Choi, Aquat. Toxicol.97, 116 (2010).

31. K. Konopka and T. Goslinski, J. Dent. Res. 86, 694 (2007).32. R. Bakalova, H. Ohba, Z. Zhelev, M Ishikawa, and Y. Baba, Nat.

biotechnol. 22, 1360 (2004).33. A. Besaratinia, S. Kim, and G. P. Pfeifer, FASEB. 22, 2379

(2008).34. A. Anas, T. Okuda, N. Kawashima, K. Nakayama, T. Itoh,

M. Ishikawa, and V. Biju, ACS Nano 3, 2419 (2010).35. A. Kumari and S. K. Yadav, Expert Opin. Drug Deliv. 8, 141

(2011).36. F. A. Derheimer and M. B. Kastan, FEBS Lett. 584, 3675 (2010).37. S. Y. Chein, Y. C. Wu, J. G. Chung, J. S. Yang, H. F. Lu, M. F.

Tsou, W. G. Wood, S. J. Kuo, and D. R. Chen, Hum. Exp. Toxicol.28, 493 (2009).

Received: 17 November 2011. Accepted: 18 November 2011.


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