Real-Time Investigation of Acute Toxicity of ZnO Nanoparticles on

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Real-Time Investigation of Acute Toxicity of ZnO Nanoparticles on Human Lung Epithelia with Hopping Probe Ion Conductance Microscopy Xi Yang,†,‡,§ Xiao Liu,†,§ Hujie Lu,† Xiaofan Zhang,† Liying Ma,† Ruiling Gao,† and Yanjun Zhang*,†,‡ †

Nanomedicine Laboratory, China National Academy of Nanotechnology & Engineering, Tianjin, China 300457 Department of Neurosurgery, Tianjin Medical University General Hospital; Tianjin Neurological Institute; Key Laboratory of Post-trauma Neuro-repair and Regeneration in Central Nervous System, Ministry of Education; Tianjin Key Laboratory of Injuries, Variations and Regeneration of Nervous System, Tianjin, People's Republic of China 300052

‡

S Supporting Information *

ABSTRACT: Recent studies have proved that zinc oxide nanoparticles (nZnO) can cause acute lung epithelial inflammation and respiratory toxicity; however, the mechanism of such acute negative effect on lung epithelia is still unclear. In this study, early responses of living human alveolar epithelial A549 cells after exposure to nZnO were investigated by noncontact hopping probe ion conductance microscopy (HPICM) that was combined with the patch-clamp technique. Continuous repetitive high-resolution HPICM scannings observed that 100 μg/mL nZnO treatment caused acute damage to A549 cell membrane within 1.5 h. Such membrane damage was reflected in a significantly elevated lactate dehydrogenase (LDH) level in cell culture medium after 3 h of nZnO exposure. The combined HPICM and patch-clamp technique can easily perform whole-cell patch-clamp recordings, which demonstated that nZnO treatment even could inhibit the activities of ion channels in A549 cells within 15 min. The HPICM technique is shown to be capable of detecting the acute toxicity of nZnO on living cells in real time and helping to elucidate the mechanism of its action.

1. INTRODUCTION Among many metal oxide nanoparticles (NPs), zinc oxide NPs (nZnO) have been proved to induce the highest toxicity in many biological systems, including bacteria, aquatic biota, higher plants, mammals, and human cell lines.1−7 With the extensive use of nZnO in various applications, including electronics, biosensors, medicine, paints, sunscreens, and cosmetics, it obtains a higher potential to be released into atmospheric environment; thus, we must pay more attention to its acute cytotoxicity in lung epithelia.6−11 Inhalation of nZnO causes acute lung epithelial inflammation in rodents.12−14 In a human inhalation study, the concentration of 2.5 mg/m3 ZnO fume (mass median aerodynamic diameter from 60 to 520 nm) for 2 h causes an acute metal fume fever syndrome at 6 h after exposure.15 Using human lung epithelial cell lines, many reports have studied the acute toxic effects of nZnO by evaluating membrane integrity and cell viability after 6−24 h of exposure.16−19 Some studies have also demonstrated that acute cytoxicity of nZnO in living lung epithelia can even be observed after 3 h of exposure.19,20 However, the mechanism of such an early stage of acute toxicological effect of nZnO remains poorly understood. Direct high-resolution repetitive investigations of the integrity of cell membrane might aid in the elucidation of the © 2011 American Chemical Society

acute toxicity of nZnO on living lung epithelia. The development of scanning probe microscopy (SPM), such as tapping-mode atomic force microscopy (AFM)21 and noncontact scanning ion conductance microscopy (SICM),22−24 allows us to study 3D cell membrane structures on a nanometer scale and in real time. A recent study comparing SICM to AFM on one and the same sample has demonstrated that noncontact SICM is more suitable for imaging soft cells at a comparable resolution.25 Moreover, backstep26−28 and standing approach29 operation modes of SICM have been developed to diminish the interaction/interference with the soft and complex surface of living cells during image acquisition. Novak and co-workers27 have recently developed a hopping probe mode of SICM hopping probe ion conductance microscopy (HPICM)to achieve long-term noncontact imaging of the complex surfaces of living cells at lateral nanometer-scale resolutions. This noncontact high-resolution HPICM allows one to monitor the dynamic membrane structural changes of living cells in response to NPs by performing reliable repetitive scannings. In addition, HPICM can be coupled with ion-channel patch clamping for functional testing,30,31 which makes it an ideal tool Received: August 12, 2011 Published: December 22, 2011 297

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Figure 1. SEM images of a typical HPICM scanning nanopipette. (A) Top view of a nanopipette. Scale bar, 200 nm. (B) Side view of the same nanopipette. Scale bar, 200 nm. 25, 50, and 100 μg/mL nZnO were first centrifuged and then filtered through a 100 nm sterile syringe filter (Pall Corporation, MI) to remove the nZnO particles. The concentrations of dissolved Zn2+ from nZnO suspensions were measured with inductively coupled plasma with atomic emission spectrometry (ICP-AES; IRIS Intrepid II, Thermo Scientific, United States). 2.5. Lactate Dehydrogenase (LDH) Release. Cell membrane damage induced by nZnO was assessed by LDH leakage into the culture medium. The activity of LDH in the medium was determined using a commercially available LDH diagnostic kit (Nanjing Jiancheng Bioengineering Institute, China). Cells were seeded in 24-well plate (BD Biosciences, United States), exposed to increasing concentrations of nZnO in DEMEM/F12 (0, 5, 25, 50, and 100 μg/mL), and incubated for 1, 2, or 3 h at 37 °C in a humid atmosphere with 5% CO2. After incubation, the medium from each well was centrifuged at 250g for 4 min. Then, 100 μL supernates were analyzed for LDH release. The optical absorbance at 440 nm was recorded in 1 cm optical path length using a Synergy 2 multimode microplate reader (Bio TeK, United States). Results were analyzed with Gen5 Reader Control and Data Analysis Software (Bio TeK). 2.6. Patch-Clamp Recording. An Axopatch 700B amplifier (Molecular Devices, United States) connected to a Digidata 1440A (Molecular Devices) was used to record whole-cell ion-channel currents.31,34 All evoked currents were sampled at 5 kHz and filtered with 1 kHz low-pass Bessel filter. Extracellular solution contained (in mM) the following: NaCl, 140; KCl, 5.4; MgCl2, 1; CaCl2, 2; HEPES, 10; and glucose, 10, pH 7.4. The electrode internal solution contained (in mM) the following: KCl, 140; MgCl2, 2; HEPES, 10; EGTA, 10; and Mg-ATP, 1, pH 7.3. Both external and internal solutions were filtered with 0.1 μm sterile syringe filters (Pall Corporation). All experiments were carried out at room temperature. 2.7. Preparation of Scanning Nanopipette and PatchClamping Micropipet. All pipettes were pulled from borosilicate glass (O.D., 1.00 mm; I.D., 0.59 mm; length, 90 mm; Vitalsense Scientific Instruments, China) using a P-2000 laser-based puller (Sutter Instruments Co., United States). For HPICM scanning, the resistances of nanopipettes ranged from 100 to 130 MΩ when they were filled with L15 medium, and their diameters were evaluated by scanning electron microscopy (SEM). Figure 1 presents SEM images of a typical HPICM nanopipette. The measured inner/outer diameters of this nanopipette were about 75/112 nm. For whole-cell patch-clamp recordings, micropipets had resistances of 2−6 MΩ when they were filled with the internal solution. 2.8. HPICM Setup. The HPICM setup was upgraded from a commercial ICnano Scanning Ion Conductance Microscope (SICM) (Ionscope Ltd., United Kingdom) as discribed previously.27,31,35 Briefly, a SH01 scan head (Ionscope Ltd.) was placed on the platform of inverted TiU microscope (Nikon Corporation, Japan). The ICnano controller (Ionscope Ltd.) controlled the vertical Z direction Piezo (25 μm, P-753.21C, Physik Instrumente, Germany) to perform positioning and hopping of the nanopipette probe and also the scanning of A549 cells under the nanopipette in the XY plane by two PIHera Piezo (100 μm, P-621.2C, Physik Instrumente). An external Axon MultiClamp 700B amplifier (Molecular Devices) was used to detect the ion current flowing into the nanopipette and provide a +200 mV DC voltage between the nanopipette electrode and the bath electrode. When a probe approached the A549 cell membrane, a reference DC current Iref was measured as an average of the ion current flowing into the HPICM probe. A sample pipet distance with 0.4% reduction of Iref was

to study acute nZnO toxicity effects on living lung epithelia in real time. Lung alveolar epithelial type II (AT II) cells perform a variety of important functions, including regulation of surfactant metabolism, ion transportation, and alveolar repairment in response to injury.32 A human bronchoalveolar carcinomaderived A549 cell line has been widely used as an in vitro AT II epithelial cell model to investigate the pulmonary toxicity of inhaled foreign materials and NPs.33 In this study, early responses of living human alveolar epithelial A549 cells after exposure to nZnO were investigated by noncontact highresolution HPICM and its combined patch-clamp technique.

2. EXPERIMENTAL PROCEDURES 2.1. Reagents. DMEM/F12 (1:1) medium was purchased from Hyclone (Logan, UT). Fetal bovine serum (FBS), L15 medium, penicillin, and streptomycin were bought from Gibco (Langley, OK). Adenosine 5′-triphosphate magnesium salt (Mg-ATP) and L-glutamine were purchased from Sigma-Aldrich (St. Louis, MO). 2.2. Cell Culture. A549 cells were purchased from American Type Culture Collection (80−100 passages). Cells were grown in DMEM/ F12 supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/ mL streptomycin at 37 °C in a 5% CO2 humidified atmosphere (series II water jacketed CO2 incubator, ThermoForma, United States). Cells were passaged once per week with medium changes every 2−3 days. For morphological analysis and electrophysiological studies, cells were plated onto 35 mm Petri dishs (Corning, United States) at a density of 1 × 104 cells/dish for 2−4 days or in 0.4 μm pore-size PET membrane filter cell culture inserts (BD Biosciences, United States) at a density of 1 × 105 cells/filter for 6−8 days. 2.3. Preparation and Characterization of nZnO. Uncoated nZnO powder with a primary size of 20 nm was purchased from Nanjing High Technology Nano Co., Ltd. (Nanjing, China). The average particle size distribution and shape of the nZnO were determined by transmission electron microscopy (TEM; JEM2010FEF, JEOL, Japan). To prepare TEM samples, the nZnO powder was dropped onto the surface of a copper grid with a carbon film at room temperature. To further analyze the physicochemical properties of nZnO powder, its surface area was measured by the Brunauer−Emmett−Teller (BET) method (Quantachrome Instruments, model AutoSORB-1), and crystallinity was determined by an X-ray diffraction (XRD) technique (Bruker-Axs, model D8 Advance). The mean size, polydispersity index (PDI), and surface charge (ζpotential) of nZnO suspension were examined by dynamic light scattering (DLS) and photon correlation spectroscopy (Malvern Instruments, model Zetasizer-3000). The PDI values range from 0 to 1, and a bigger PDI value indicates a less homogeneous size distribution of nZnO. DLS measurements were taken on nZnO in DMEM/F12 cell culture media. A stock solution (10 mg/mL) of nZnO was prepared in ultrapure water and dispersed by a 400 W sonicator (UP400s, Dr. Hielscher GmbH, Germany) for 30 min. In each study, the suspension was freshly prepared and then diluted to the specified concentrations with DMEM/F12 medium for the experiments. 2.4. Zinc Ion Release of nZnO in Cell Culture Medium. To quantify the dissolved zinc ion in DMEM/F12 medium, solutions of 5, 298

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Figure 2. Characterization of nZnO. (A) TEM image of nZnO powder. (B) XRD pattern of nZnO powder. set to maintain the distance between the nanopipette and the A549 cell, and the Z-position of probe was recorded as the height of A549 cell at this XY scanning point, which was used to generate a topographical image of A549 cell. The time required to scan a big area of 80 μm × 80 μm or 90 μm × 90 μm with a typical resolution of 128 × 128 pixels was about 20−30 min. 2.9. Image Processing and Data Analysis. The raw topography data were processed and analyzed with SICM Image Viewer software (Ionscope Ltd.). All patch-clamp recordings were analyzed with Clampfit 10.2 (Axon Instruments, United States) and Microcal Origin software version 8.0 (Microcal Software Inc., United States). Values were presented as means ± SEMs. Statistical significance was determined by Student's t test. P values less than 0.05 were considered to be statistically significant.

36-1451) of International Centre for Diffraction Data (United States). The crystallite size of nZnO was estimated as about 30 nm using the full width at half-maximum peak height of the XRD signature (Table 1). The surface area of nZnO was measured as 44.7 m2/g by the BET method (Table 1). The nZnO particles tended to form agglomerate in the cell culture medium.18,19 The hydrodynamic size distribution and surface charges of nZnO were inspected with DLS and laser Doppler electrophoresis (Table 1). When dispersed in the DMEM/F12 medium, the average mean particle size of nZnO was about 246.9 nm. Their PDI value and surface charges were measured as 0.44 and −12.6 mV, respectively. 3.2. Acute nZnO Cytotoxicity in A549 Cells. To study the acute cytotoxic efftects of nZnO, nZnO-induced cell morphology changes in living A549 cells were first observed with phase-contrast optical microscopy (Figure S1 in the Supporting Information). After 2 h of exposure, nZnO treatment did not cause any visible cell damage at lower concentration but caused a portion of A549 cells to become rounded in cell morphology at a concentration of 100 μg/mL (Figure S1A in the Supporting Information). Continuous optical microscopy observations were used to follow the early responses of cells incubated with 100 μg/mL nZnO (Figure S1B in the Supporting Information), but these low-resolution images can neither distinguish the cytotoxicity earlier than 2 h nor reveal the membrane changes in fine details. High-resolution HPICM scannings were then performed to reveal the membrane structure changes in response to 2 h of 100 μg/mL nZnO treatment (Figure 3). As compared with untreated A549 cells (Figure 3A), treatment with nZnO caused severe membrane deformations in the rounded A549 cells (Figure 3B,C). Possible apoptotic bodies, which had been

3. RESULTS 3.1. Characterization of nZnO. Different physicochemical properties of nZnO were observed (Figure 2 and Table 1). The Table 1. Physicochemical Properties of nZnO

sample

crystallite size in dry state by XRD

BET surface area

average mean size in DMEM/ F12

polydispersity index (PDI)

surface charge

ZnO

30 nm

4.7 m2/g

246.9 nm

0.44

12.6 mV

TEM image shows that hexagonal-shaped nZnO powders have variable sizes ranging from 20 to 50 nm (Figure 2A). The crystalline phase structure of nZnO was depicted in the X-ray diffraction (XRD) spectrum (Figure 2B). The diffraction peaks corresponding to ZnO with hexagonal type XRD pattern were detected and verified with the standard data file (PDF number

Figure 3. HPICM topological images of one untreated control (A) and two rounded nZnO-treated (B and C) A549 cells; “ab” represents the possible apoptotic body. 299

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previously detected with AFM from apoptotic A549 cells,36 were also observed in these rounded A549 cells (Figure 3, “ab” = apoptotic body). 3.3. LDH Release from nZnO-Treated A549 Cells. Release of LDH to the cell culture medium indicates nZnOinduced membrane damage of A549 cell (Figure 4). There was

in living A549 cells within 3 h of exposure. As compared with untreated control cell (Figure 5A), repetitive HPICM scannings demonstrated that nZnO treatment began to cause acute damage to the A549 cell membrane after 1 h of exposure (Figure 5B,C). Some “holelike” membrane damages were observed from 1 h nZnO-treated A549 cell (Figure 5B, 1 h frame), and such membrane damages were aggravated following the exposure time (note the increasing black dots in Figure 5B, 1−2 h frames). Figure 5C presents four images chosen from another series of time-lapse HPICM scans and demonstrates another kind of acute toxical effect of nZnO on cell membrane deformations. As compared with control cell (Figure 5C, control frame), the 1 h nZnO-treated A549 cell had some filopodia structures around the rounded cell body, whose structures were more and more obvious following the increasing exposure time with nZnO (Figure 5C, 1−4 h frames). 3.5. High-Resolution Investigation of Acute nZnO Toxicity on Cell Membrane Deformation. To observe the details of deformed cell membrane induced by nZnO, further high-resolution HPICM observations were performed (Figure 6). Figure 6A presents one HPICM image of an untreated A549 cell. The white-dotted square marked region of cell surface was further scanned with even higher resolution (Figure 6B). It showed that the untreated A549 cell membrane was intact and covered with visible microvilli (Figure 6B). The HPICM scanning profile of the dotted line indicated clearly these outward microvilli membrane protrusions (asterisks highlighted in Figure 6C). Exposure of this A549 cell to 100 μg/mL nZnO for 1.5 h and the inward “holelike” membrane damage were clearly observed (Figure 6D, note the whitedotted circle highlighted area for comparison). A further highresolution zoom-in HPICM scanning of nZnO-treated A549 cell indicated more details of the cell membrane damages with microvilli disappearance and “holelike” membrane structures appearance (Figure 6E). The HPICM scanning profile of the dotted line presented more details of these inward “holelike” membrane damages (marked with letters a−c in Figure 6F).

Figure 4. LDH release from A549 cells after exposed to different concentrations of nZnO for 1−3 h. Values are presented as means ± SEMs (n = 6). An asterisk denotes significance in comparison to control values (P < 0.05).

no significant difference between the mean values of LDH release from A549 cells exposed to various doses of nZnO for 2 h when compared with the values of the control cells. LDH levels were only significantly increased (p < 0.05, n = 6) after 3 h of exposure to 50 and 100 μg/mL nZnO (Figure 4). 3.4. Continuous HPICM Observations of Acute nZnO Cytotoxicity in A549 Cells. According to our observations and the previous published results by other groups,7,17 a concentration of 100 μg/mL was chosen in our following experiments to ensure an effective acute cytotoxicity of nZnO

Figure 5. Continuous HPICM topological scannings of control and nZnO-treated A549 cells. (A) Four HPICM topological images chosen from a series of time-lapse scans of an untreated A549 cell in DMEM/F12 medium for 4 h as a control experiment. (B and C) Two series of continuous HPICM scannings of acute nZnO cytotoxic effects on living A549 cells in DMEM/F12 medium. 300

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Figure 6. High-resolution HPICM scannings of acute nZnO cytotoxic effects on living A549 cells in DMEM/F12 medium. (A) HPICM image of a typical A549 cell (90 μm × 90 μm). (B) A 10 μm × 10 μm high-resolution zoom-in HPICM image of A549 cell membrane from the white-dotted square marked area in part A; the “asterisk” represents microvillus structure. (C) The scanning profile of the white-dotted line marked in part B. (D) HPICM image of same A549 cell, which was treated with 100 μg/mL nZnO for 1.5 h (90 μm × 90 μm). (E) High-resolution zoom-in HPICM image of nZnO-treated A549 cell membrane from the white-dotted square marked area in part D. The a−c markers indicate the “holelike” membrane deformations. (F) The scanning profile of white-dotted line marked in part E.

Figure 7. Four HPICM images chosen from a series of 4 h continuous repetitive scannings of 10 μg/mL ZnCl2-treated A549 cell.

Figure 8. Twelve hour continuous repetitive HPICM scannings of a nZnO-treated confluent A549 cell monolayer in CO2-independent L15 medium. The arrowheads highlight the “holelike” membrane damages of indivadual A549 cells. The white-dotted circles indicate the filopodia and the gap structure between A549 cells in epithelial monolayer.

3.6. Role of Dissolved Zn2+ in Acute nZnO Cytotoxicity in A549 Cells. Uncoated nZnO is slightly soluble and can release Zn2+ in cell culture medium, and the dissolved Zn2+ is considered to account for the toxic effects of nZnO on living cells.37−39 In this study, we observed that nZnO caused acute cell membrane damages to A549 cells, but the relevance of the

dissolved Zn2+ to these negative effects was unclear. Under the help of ICP-AES, we measured the dissolved Zn2+ in DMEM/ F12 for different concentrations of nZnO (Figure S2 in the Supporting Information). The dissolved Zn2+ from 100 μg/mL nZnO in DMEM/F12 for 4 h was about 10 μg/mL, whose value was similar to the result published by other group.38 301

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Figure 9. Acute effects of nZnO on the activities of ion channels in A549 cells. (A) Representative recordings show activation of the whole-cell currents under control conditions. (B) Representative recordings show rapid inhibition of the whole-cell currents by 100 μg/mL nZnO within 15 min. (C) Current−voltage relationships correspond to 20 recordings obtained from untreated cells and 11 recordings from nZnO-treated cells. Asterisks indicate values statistically different from the control experiments (* P < 0.05). (D) Voltage protocols used to obtain the current−voltage relationships.

To study the effects of dissolved Zn2+ on living A549 cells, their membrane morphological changes in response to 10 μg/ mL ZnCl2 were investigated with repetitive HPICM scannings (Figure 7). The same as the HPICM observations obtained from those control cells in DMEM/F12 (Figure 5A), 10 μg/mL Zn2+ treatment did not cause any obvious morphological changes in A549 cells within 4 h (Figure 7). 3.7. Acute Cytotoxicity Effects of nZnO on A549 Cell Monolayer. A549 cells formed a confluent monolayer when they were cultured on membrane filter inserts for 6−8 days. To assess the toxicologic effects of 100 μg/mL nZnO on the morphology and integrity of the A549 cell monolayer, topographical images of nZnO-treated cell monolayer were studied with continuous HPICM scannings (Figure 8). As compared with the HPICM image of untreated A549 cell monolayer (Figure 8, control 0 h), some “holelike” membrane damages were presented in A549 cells membrane after 4 h of nZnO treatment (arrowheads indicated in Figure 8, +nZnO 4 h frame). Following a longer exposure time, individual A549 cells were shrinking with the expansion of intercellular space (whitedotted circle marked in Figure 8, +nZnO 8 h frame), which caused the loss of integrity of the A549 cell monolayer (Figure 8, +nZnO 12 h frame). 3.8. Acute Effect of nZnO on Ion-Channel Activities of A549 Cells. Under the help of HPICM combined patch-clamp technique, whole-cell patch-clamp recordings from nZnOtreated cells were performed (Figure 9). A representative whole-cell recording obtained from the untreated A549 cell was presented in Figure 9A. As compared with untreated cells, nZnO-treated A549 cells had lower whole-cell currents (Figure 9B). Upon the application of nZnO, such inhibitory effects reached a peak value within 15 min (p < 0.05, n = 11). The current−voltage (I−V) relationships for these recordings are shown in Figure 8C. In the presence of nZnO, the amplitude of total current (pA/pF) was significantly decreased at the test potentials between −10 and +60 mV. These whole-cell currents were consistently evoked by applying 10 mV steps of 450 ms duration from a hold potential of −70 to +60 mV (Figure 9D).

4. DISCUSSION Assessing the safety of synthetic NPs has currently become a worldwide issue, and several studies have focused on the cytotoxicity and respiratory toxicity of various types of NPs.6,7,19,40 It has been known that ∼45% of 10 nm, ∼50% of 20 nm, and ∼25% of 100 nm NPs can deposit in the alveoli.11 Once ZnO NPs are taken up by alveolar cells, they are likely to induce acute toxic effects and damage lung epithelia through a so far unclear mechanism.16,18,19,37,38,41 Among these cytotoxicity studies, the most commonly used methods are endpoint assays from those nZnO-treated cells after a few hours of exposure, and the real-time noninvasive high-resolution investigation of the early responses of living lung epithelia to nZnO is still missing. With the help of the recently developed noncontact highresolution HPICM and its combined patch-clamp technique, we continuously investigated the early responses of living alveolar epithelial A549 cells after exposure to nZnO in this study. Repetitive HPICM topological scannings demonstrated that 100 μg/mL nZnO treatment caused membrane damage in A549 cells after 1 h of exposure, and this damage was aggravated in a time-dependent manner. Such HPICM observed membrane integrity lost of A549 cells was later gauged by a significant increase in LDH release in medium after 3 h of nZnO exposure. After 4 h of incubation with nZnO, “holelike” membrane damage of individual cells was also observed in the A549 cell monolayer, and the adhesive coupling between adjacent cells was disrupted gradually following the increasing exposure time with nZnO. In addition, whole-cell patch-clamp recordings showed that nZnO treatment inhibited the activities of ion channels in A549 cells within 15 min. All of these data strongly suggest the potential acute adverse health outcomes of high-concentration nZnO. There were suggestions that nZnO cytotoxicity in lung epithelia could be affected by the release of Zn2+ through the dissolution of nZnO.37,38 In this study, we measured that the liberated Zn2+ from 100 μg/mL nZnO was only about 10 μg/ mL after 4 h of exposure. Adding such an amount of Zn2+ did not cause any A549 cell morphology changes during 4 h HPICM observations. This indicated that our observed acute 302

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(9) Nel, A., Xia, T., Mädler, L., and Li, N. (2006) Toxic potential of materials at the nanolevel. Science. 311, 622−627 [PubMed: 16456071]. (10) Geiser, M., and Kreyling, W. G. (2010) Deposition and biokinetics of inhaled nanoparticles. Part. Fibre Toxicol. 7, 2−17 [PubMed: 20205860]. (11) Yokel1, R. A., and MacPhail, R. C. (2011) Engineered nanomaterials: Exposures, hazards, and risk prevention. J. Occup. Med. Toxicol. 6, 7[PubMed: 21418643]. (12) Conner, M. W., Flood, W. H., Rogers, A. E., and Amdur, M. O. (1988) Lung injury in guinea pigs caused by multiple exposures to ultrafine zinc oxide: changes in pulmonary lavage fluid. J. Toxicol. Environ. Health 25, 57−69 [PubMed: 3418745]. (13) Lam, H. F., Chen, L. C., Ainsworth, D., Peoples, S., and Amdur, M. O. (1988) Pulmonary function of guinea pigs exposed to freshly generated ultrafine zinc oxide with and without spike concentrations. Am. Ind. Hyg. Assoc. J. 49, 333−341 [PubMed: 3407592]. (14) Wesselkamper, S. C., Chen, L. C., and Gordon, T. (2001) Development of pulmonary tolerance in mice exposed to zinc oxide fumes. Toxicol. Sci. 60, 144−151 [PubMed: 11222881]. (15) Fine, J. M., Gordon, T., Chen, L. C., Kinney, P., Falcone, G., and Beckett, W. S. (1997) Metal fume fever: characterization of clinical and plasma IL-6 responses in controlled human exposures to zinc oxide fume at and below the threshold limit value. J. Occup. Environ. Med. 39, 722−726 [PubMed: 9273875]. (16) Huang, C. C., Aronstam, R. S., Chen, D. R., and Huang, Y. W. (2010) Oxidative stress, calcium homeostasis, and altered gene expression in human lung epithelial cells exposed to ZnO nanoparticles. Toxicol. in Vitro 24, 45−55 [PubMed: 19755143]. (17) Xu, M., Fujita, D., Kajiwara, S., Minowa, T., Li, X., Takemura, T., Iwai, H., and Hanagata, N. (2010) Contribution of physicochemical characteristics of nano-oxides to cytotoxicity. Biomaterials 31, 8022− 8031 [PubMed: 20688385]. (18) Hsiao, I. L., and Huang, Y. J. (2011) Effects of various physicochemical characteristics on the toxicities of ZnO and TiO2 nanoparticles toward human lung epithelial cells. Sci. Total Environ. 409, 1219−1228 [PubMed: 21255821]. (19) Lanone, S., Rogerieux, F., Geys, J., Dupont, A., MaillotMarechal, E., Boczkowski, J., Lacroix, G., and Hoet, P. (2009) Comparative toxicity of 24 manufactured nanoparticles in human alveolar epithelial and macrophage cell lines. Part. Fibre Toxicol. 6, 14[PubMed:19405955]. (20) George, S., Pokhrel, S., Xia, T., Gilbert, B., Ji, Z., Schowalter, M., Rosenauer, A., Damoiseaux, R., Bradley, K. A., Mädler, L, and Nel, A. E. (2010) Use of a rapid cytotoxicity screening approach to engineer a safer zinc oxide nanoparticle through iron doping. ACS Nano 4, 15−29 [PubMed: 20043640]. (21) Allison, D. P., Mortensen, N. P., Sullivan, C. J., and Doktycz, M. J. (2010) Atomic force microscopy of biological samples. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2, 618−634 [PubMed: 20672388]. (22) Hansma, P. K., Drake, B., Marti, O., Gould, S. A., and Prater, C. B. (1989) The scanning ion-conductance microscope. Science 243, 641−643 [PubMed: 2464851]. (23) Korchev, Y. E., Bashford, C. L., Milovanovic, M., Vodyanoy, I., and Lab, M. J. (1997) Scanning ion conductance microscopy of living cells. Biophys. J. 73, 653−658 [PubMed: 9251784]. (24) Zhang, Y., Gorelik, J., Sanchez, D., Shevchuk, A., Lab, M., Vodyanoy, I., Klenerman, D., Edwards, C., and Korchev, Y. (2005) Scanning ion conductance microscopy reveals how a functional renal epithelial monolayer maintains its integrity. Kidney Int. 68, 1071−1077 [PubMed: 16105037]. (25) Rheinlaender, J., Geisse, N. A., Proksch, R., and Schäffer, T. E. (2011) Comparison of scanning ion conductance microscopy with atomic force microscopy for cell imaging. Langmuir 27, 697−704 [PubMed:21158392]. (26) Mann, S. A., Hoffmann, G., Hengstenberg, A., Schuhmann, W., and Dietzel, I. D. (2002) Pulse-mode scanning ion conductance

cytotoxicity of nZnO in A549 cells was independent of the Zn2+; however, we could not rule out the accumulated toxicity of these released Zn2+ on A549 cells for a long-term nZnO exposure. Although the involved intracellular biochemical mechanisms and ion channels in our observed acute nZnO toxicity remain to be determined, this study provides direct evidence that 100 μg/mL nZnO can cause acute membrane damage to alveolar epithelia after 1 h of exposure. Such acute cytotoxicity of nZnO in A549 cells may be mainly associated with the early membrane damage. The noncontact HPICM technique is demonstrated to be suitable for high-resolution investigation of the acute morphological changes of living cells in response to NPs in real time.

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ASSOCIATED CONTENT

S Supporting Information *

Phase-contrast optical microscopy images of nZnO-treated A549 cells in DMEM/F12 medium (Figure S1) and the dissolved Zn2+ concentration from nZnO (Figure S2). This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 86-22-62002900-1139. Fax: 86-22-62002984. E-mail: [email protected]. Author Contributions §

Both authors contributed equally to this work.

Funding

This work was supported by the National Natural Science Foundation of China (no. 30971184).

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REFERENCES

(1) Wang, B., Feng, W. Y., Wang, T. C., Jia, G., Wang, M., Shi, J. W., Zhang, F., Zhao, Y. L., and Chai, Z. F. (2006) Acute toxicity of nanoand micro-scale zinc powder in healthy adult mice. Toxicol. Lett. 161, 115−123 [PubMed: 16165331]. (2) Reddy, K. M., Feris, K., Bell, J., Wingett, D. G., Hanley, C., and Punnoose, A. (2007) Selective toxicity of zinc oxide nanoparticles to prokaryotic and eukaryotic systems. Appl. Phys. Lett. 90, 2139021− 2139023 [PubMed: 18160937]. (3) Huang, Z., Zheng, X., Yan, D., Yin, G., Liao, X., Kang, Y., Yao, Y., Huang, D., and Hao, B. (2008) Toxicological effect of ZnO nanoparticles based on bacteria. Langmuir 24, 4140−4144 [PubMed:18341364]. (4) Lin, D., and Xing, B. (2008) Root uptake and phytotoxicity of ZnO nanoparticles. Environ. Sci. Technol. 42, 5580−5585 [PubMed: 18754479]. (5) Aruoja, V., Dubourguier, H. C., Kasemets, K., and Kahru, A. (2009) Toxicity of nanoparticles of CuO, ZnO and TiO2 to microalgae Pseudokirchneriella subcapitata. Sci. Total Environ. 407, 1461−1468 [PubMed:19038417]. (6) Hu, X., Cook, S., Wang, P., and Hwang, H. M. (2009) In vitro evaluation of cytotoxicity of engineered metal oxide nanoparticles. Sci. Total Environ. 407, 3070−3072 [PubMed: 19215968]. (7) Kim, I. S., Baek, M., and Choi, S. J. (2010) Comparative cytotoxicity of Al2O3, CeO2, TiO2 and ZnO nanoparticles to human lung cells. J. Nanosci. Nanotechnol. 10, 3453−3458 [PubMed: 20358977]. (8) Geiser, M., Rothen-Rutishauser, B., Kapp, N., Schürch, S., Kreyling, W., Schulz, H., Semmler, M., Im Hof, V., Heyder, J., and Gehr, P. (2005) Ultrafine particles cross cellular membranes by nonphagocytic mechanisms in lungs and in cultured cells. Environ. Health Perspect. 113, 1555−1560 [PubMed:16263511]. 303

dx.doi.org/10.1021/tx2004823 | Chem. Res. Toxicol. 2012, 25, 297−304

Chemical Research in Toxicology

Article

microscopyA method to investigate cultured hippocampal cells. J. Neurosci. Methods 116, 113−117 [PubMed: 12044660]. (27) Novak, P., Li, C., Shevchuk, A. I., Stepanyan, R., Caldwell, M., Hughes, S., Smart, T. G., Gorelik, J., Ostanin, V. P., Lab, M. J., Moss, G. W., Frolenkov, G. I., Klenerman, D., and Korchev, Y. E. (2009) Nanoscale live-cell imaging using hopping probe ion conductance microscopy. Nature Methods 6, 279−281 [PubMed: 19252505]. (28) Happel, P., and Dietzel, I. D. (2009) Backstep scanning ion conductance microscopy as a tool for long term investigation of single living cells. J. Nanobiotechnol. 7, 7[PubMed:19860879]. (29) Takahashi, Y., Murakami, Y., Nagamine, K., Shiku, H., Aoyagi, S., Yasukawa, T., Kanzaki, M., and Matsue, T. (2010) Topographic imaging of convoluted surface of live cells by scanning ion conductance microscopy in a standing approach mode. Phys. Chem. Chem. Phys. 12, 10012−10017 [PubMed: 20485766]. (30) Gorelik, J., Gu, Y., Spohr, H. A., Shevchuk, A. I., Lab, M. J., Harding, S. E., Edwards, C. R., Whitaker, M., Moss, G. W., Benton, D. C., Sánchez, D., Darszon, A., Vodyanoy, I., Klenerman, D., and Korchev, Y. E. (2002) Ion channels in small cells and subcellular structures can be studied with a smart patch-clamp system. Biophys. J. 83, 3296−3303 [PubMed: 12496097]. (31) Yang, X., Liu, X., Zhang, X., Lu, H., Zhang, J., and Zhang, Y. (2011) Investigation of morphological and functional changes during neuronal differentiation of PC12 cells by combined Hopping Probe Ion Conductance Microscopy and patch-clamp technique. Ultramicroscopy 111 (8), 1417−1422 [PubMed: 21864785]. (32) Dobbs, L. G. (1990) Isolation and culture of alveolar type II cells. Am. J. Physiol. 258, L134−147 [PubMed:2185652]. (33) Lieber, M., Smith, B., Szakal, A., Nelson-Rees, W., and Todaro, G. (1976) A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells. Int. J. Cancer 17, 62− 70 [PubMed: 175022]. (34) Hamill, O. P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F. J. (1981) Improved patch clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 391, 85−100 [PubMed:6270629]. (35) Liu, X., Yang, X., Zhang, B., Zhang, X., Lu, H., Zhang, J., and Zhang, Y. (2011) High-resolution morphological identification and characterization of living neuroblastoma SK-N-SH cells by hopping probe ion conductance microscopy. Brain Res. 1386, 35−40 [PubMed: 21354113]. (36) Tomankova, K., Kolarova, H., Bajgar, R., Jirova, D., Kejlova, K., and Mosinger, J. (2009) Study of the photodynamic effect on the A549 cell line by atomic force microscopy and the influence of green tea extract on the production of reactive oxygen species. Ann. N.Y. Acad. Sci. 1171, 549−558 [PubMed: 19723103]. (37) Xia, T., Kovochich, M., Liong, M., Mädler, L., Gilbert, B., Shi, H., Yeh, J. I., Zink, J. I., and Nel, A. E. (2008) Comparison of the mechanism of toxicity of zinc oxide and cerium oxide nanoparticles based on dissolution and oxidative stress properties. ACS Nano 2, 2121−2134 [PubMed: 19206459]. (38) Song, W., Zhang, J., Guo, J., Zhang, J., Ding, F., Li, L., and Sun, Z. (2010) Role of the dissolved zinc ion and reactive oxygen species in cytotoxicity of ZnO nanoparticles. Toxicol. Lett. 199, 389−397 [PubMed: 20934491]. (39) Deng, X., Luan, Q., Chen, W., Wang, Y., Wu, M., Zhang, H., and Jiao, Z. (2009) Nanosized zinc oxide particles induce neural stem cell apoptosis. Nanotechnology 20, 115−101 [PubMed: 19420431]. (40) Lewinski, N., Colvin, V., and Drezek, R. (2008) Cytotoxicity of nanoparticles. Small 4, 26−49 [PubMed: 18165959]. (41) Heng, B. C., Zhao, X., Xiong, S., Ng, K. W., Boey, F. Y., and Loo, J. S. (2010) Toxicity of zinc oxide (ZnO) nanoparticles on human bronchial epithelial cells (BEAS-2B) is accentuated by oxidative stress. Food Chem. Toxicol. 48, 1762−1766 [PubMed: 20412830].

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dx.doi.org/10.1021/tx2004823 | Chem. Res. Toxicol. 2012, 25, 297−304