Chemosphere 135 (2015) 240–249

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Necrotic cell death induced by the protein-mediated intercellular uptake of CdTe quantum dots Lu Lai a,b, Jian-Cheng Jin a, Zi-Qiang Xu a, Ping Mei b, Feng-Lei Jiang a,⇑, Yi Liu a,c,⇑ a State Key Laboratory of Virology & Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecule Sciences, Wuhan University, Wuhan 430072, PR China b College of Chemistry and Environmental Engineering, Yangtze University, Jingzhou 434023, PR China c College of Chemistry and Material Sciences, Hubei Engineering University, Xiaogan 432000, PR China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 MPT is an important step in QDs-

induced necrosis.  QDs induce MPT through the

elevation of ROS.  The adsorption of serum protein will

mediate the cellular uptake of QDs.  The rapid degradation of QDs in

lysosome induces cell necrosis.

a r t i c l e

i n f o

Article history: Received 25 December 2014 Received in revised form 6 April 2015 Accepted 17 April 2015

Handling Editor: Tamara S. Galloway Keywords: Reactive oxygen species Mitochondrial permeability transition Necrosis Internalization of QDs

a b s t r a c t The toxicity of CdTe QDs with nearly identical maximum emission wavelength but modified with four different ligands (MPA, NAC, GSH and dBSA) to HEK293 and HeLa cells were investigated using flow cytometry, spectroscopic and microscopic methods. The results showed that the cytotoxicity of QDs increased in a dose- and time-dependent manner. No appreciable fraction of cells with sub-G1 DNA content, the loss of membrane integrity, and the swelling of nuclei clearly indicated that CdTe QDs could lead to necrotic cell death in HEK293 cells. JC-1 staining and TEM images confirmed that QDs induced MPT, which resulted in mitochondrial swelling, collapse of the membrane potential. MPT is an important step in QDs-induced necrosis. Moreover, QDs induced MPT through the elevation of ROS. The fluorimetric assay and theoretical analysis demonstrated ROS production has been associated with the internalization of QDs with cells. Due to large surface/volume ratios of QDs, when QDs added in the culture medium, serum proteins in the culture medium will be adsorbed on the surface of QDs. This adsorption of serum protein will change the surface properties and size, and then mediate the cellular uptake of QDs via the clathrin-mediated endocytic pathway. After entering into cells, the translocation of QDs in cells is usually via endosomal or lysosomal vesicles. The rapid degradation of QDs in lysosome and the lysosomal destabilization induce cell necrosis. This study provides a basis for understanding the cytotoxicity mechanism of CdTe QDs, and valuable information for safe use of QDs in the future. Ó 2015 Elsevier Ltd. All rights reserved.

Abbreviations: MPA, 3-Mercaptopropionic acid; NAC, N-acetyl-L-cysteine; GSH, L-glutathione reduced; dBSA, denatured bovine serum albumin; QDs, quantum dots; MPT, mitochondrial permeability transition; ROS, reactive oxygen species; HEK293, human embryonic kidney 293 cells; TEM, transmission electron microscopy. ⇑ Corresponding authors at: State Key Laboratory of Virology & Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecule Sciences, Wuhan University, Wuhan 430072, PR China (Y. Liu). Tel.: +86 27 6875 6667; fax: +86 27 6875 4067. E-mail addresses: fl[email protected] (F.-L. Jiang), [email protected] (Y. Liu). http://dx.doi.org/10.1016/j.chemosphere.2015.04.044 0045-6535/Ó 2015 Elsevier Ltd. All rights reserved.

L. Lai et al. / Chemosphere 135 (2015) 240–249

1. Introduction Quantum dots are tiny particles, or ‘‘nanoparticles’’, range from 2 to 10 nanometers in diameter (about the width of 50 atoms). Because of their small size, quantum dots display unique optical and electrical properties that are different from those of the corresponding bulk material (Murray et al., 2000). The wavelength of these photon emissions do not depend on the composition of the material, but its size (Ekimov and Onushchenko, 1981). During production, quantum dots can therefore be ‘‘tuned’’ to emit any color of light desired (Leutwyler et al., 1996). Researchers have studied the applications of quantum dots in solar cells (Nozik, 2002), LEDs (Leutwyler et al., 1996), diode lasers (Narukawa et al., 1997), medical imaging (Michalet et al., 2005), and quantum computing (Loss and DiVincenzo, 1998). However, the most optically suitable emitting materials were cadmium-based materials. To prevent potential threats originating from accidental exposure of quantum dots, their toxicity has been extensively studied at the levels of biological macromolecules (Atay et al., 2010; Vannoy and Leblanc, 2010; Xiao et al., 2012), subcellular organelles (Li et al., 2011), cell (Derfus et al., 2004; Hoshino et al., 2004; Lu et al., 2008; Chibli et al., 2011; Clift et al., 2011; Neibert and Maysinger, 2012), protozoa (Werlin et al., 2011), and others (Donaldson et al., 2002; Mortensen et al., 2013; Yang et al., 2014). Nanotoxicology is an important subdiscipline of nanotechnology that investigates the interaction between nanomaterial and biological systems (Oberdorster et al., 2005). To date, the majority of nanotoxicity research focused on the cell culture system. The extent of cytotoxicity has been found to be dependent upon composition, size, shape, concentration of QDs, surface chemistry, capping materials, etc (Hoshino et al., 2004; Lovric´ et al., 2005a,b; Hardman, 2006). Simultaneously, various mechanisms have been speculated to be responsible for QDs cytotoxicity. It is widely accepted that the release of heavy metal ion and generation of reactive oxygen species (ROS) caused by highly reactive atoms at the particle surface are important mechanisms in the cytotoxic effects of quantum dots (Derfus et al., 2004; Chibli et al., 2011). Derfus et al. indicated the cytotoxicity of QDs correlates with the liberation of free Cd2+ ions (Derfus et al., 2004). Subsequently, Su et al. further investigated the relationship between the cytotoxicity of QDs and free Cd ions (Su et al., 2010). They found that the cytotoxicity of CdTe QDs cannot be attributed solely to the toxic effect of free Cd2+, but also based on the concentration of total QDs ingested by cells. Additionally, Lovric et al. indicated that QDs-induced cell death is not classical apoptosis and QDs-induced ROS caused the damage to the plasma membrane, mitochondrion and nucleus (Lovric´ et al., 2005a,b). However, Yan et al. made a contradictory statements that CdTe QDs induced endothelial toxicity through activation of mitochondrial death pathway and induction of endothelial apoptosis, nevertheless they also admitted that ROS play an important role in mediating QDs-induced cytotoxicity (Yan et al., 2011). More recently, studies have shown that the cytotoxicity of QDs may arise from the intracellular uptake of QDs (Bottrill and Green, 2011; Gomes et al., 2011; Chen et al., 2012). Cell membrane is a selective membrane, isolates cells from the external environment and serves as a permeability barrier to prevent the entry of toxic compounds but allows the influx of nutrient molecules. It is reported that engineered nanoparticles such as gold nanoparticles, QDs, fullerene and carbon dots could pass through cell membrane and transport into cells (Sharifi et al., 2012). The known cellular uptake pathway mainly include caveolae-dependent or clathrinmediated endocytosis, phagocytosis and pinocytosis (Zhao et al.,

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2011). After enter into cells, nanoparticles are usually captured by endosomal or lysosomal vesicles of an acidic and oxidative environments, which may induce the destruction of the internalized nanoparticles (Zhao et al., 2011). Therefore, the cytotoxicity of QDs may arise from the cellular degradation of QDs. So far, most previous studies always pay attention to examine the extracellular exposure of QDs. Considering this, urgently required to examine whether cellular uptake, location and degradation are principal reasons for QDs cytotoxicity. In order to stabilize nanoparticles, alleviate toxicity and target organs, quantum dots are usually coated with ligands, polymers or functionalized with peptide sequences. It is widely accepted that the extent of QDs cytotoxicity is related to their surface chemistry (Yang et al., 2009). Furthermore, surface chemistry of QDs will change their cellular uptake pathway (Hoshino et al., 2004). However, the size or surface chemistry of QDs will change when added in the culture media and this effect cannot be neglected. We wonder if significant difference in the cytotoxicity will be observed when cells are treated with QDs modified with different small ligands. Herein, the toxicity of CdTe QDs with nearly identical maximum emission wavelength but modified with four different ligands (MPA, NAC, GSH and dBSA) to HEK293 and HeLa cells were investigated using flow cytometry, spectroscopic and microscopic methods. Our study provides a basis for understanding the mechanism of the cytotoxicity of CdTe QDs, and valuable information for safe use of QDs in the future.

2. Experimental 2.1. Cells and reagents CdCl2 (99.99%), NaBH4 (99%), tellurium powder (99.999%, about 200 mesh), bovine serum albumin (BSA), 3-Mercaptopropionic acid (MPA, 99%), N-acetyl-L-cysteine (NAC, 99%), L-glutathione reduced (GSH, 98%), and chlorpromazine hydrochloride (P98%) were obtained from Sigma–Aldrich and were used without further purification. Propidium iodide (PI), Triton X-100 (99.0%), penicillin (1650U mg 1), streptomycin (Potency (Dry basis) > 650– 850 mcg mg 1), RNase A (Type 1-A, P60%, 50–100 kU/mg 1 protein) were purchased from Biosharp Co.. Hoechst 33342 were purchased from Invitrogen Corp.. Lyso-Tracker Red were purchased from Beyotime Institute of Biotechnology. Dulbecco’s modified Eagle’s medium (DMEM) and Reduced Serum Media (Opti-MEM) were purchased from Gibco. Fetal bovine serum (FBS) were purchased from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. (China). 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Aladdin (China). Annexin V-PE apoptosis kit were obtained from eBioscience (San Diego, CA). JC-1 Mitochondrial Membrane Potential Detection Kit were purchased from MultiSciences Biotech Co., Ltd. (China). Cellular Reactive Oxygen Species detection kit (Red fluorescence, GMS 10074.1) were purchased from Genmed (USA). All other reagents were of analytical reagent grade. Ultrapure water with 18.2 MX cm (Millipore Simplicity) was used in all syntheses.

2.2. Preparation and characterization of CdTe QDs Synthesis and characterization of CdTe QDs modified with MPA, NAC, GSH and dBSA-CdTe were described in Supplementary materials. QDs concentrations were quantified using the extinction coefficients (Yu et al., 2003).

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2.3. Cell culture Human embryonic kidney cells (HEK293 cell) were obtained from Prof. Han-Zhong Wang’s research group (Wuhan Institute of Virology, Chinese Academy of Sciences). HeLa cells were obtained from Prof. Xiang Zhou’s research group (Wuhan University). HEK293 and HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 10 mg mL 1 streptomycin and 6 mg mL 1 penicillin, and maintained in the humidified atmosphere with 5% CO2 at 37 °C. For flow cytometry, HEK293 cells were seeded at a density of 3.0  105 cells/well in 6-well plate (Corning Incorporated, Corning, NY, US). 2.4. Assessment of cell metabolic activity Colorimetric 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assays were performed to assess the metabolic activity of cells. The cells in culture medium were dispensed in 96-well plate (200 lL in each well containing 5  103 HeLa cells or 4  104 HEK293 cells) and incubated overnight. If cells are attached to culture vessel growth surface, the culture media is removed and disposed. Then different amounts of QDs were dissolved in culture medium and added to each well. After 48 h treatment, medium was removed and replaced by phosphate buffer (180 lL well 1). To each well 20 lL stock MTT (5 mg mL 1) was added, and cells were then incubated for 4 h at 37 °C. Buffer was removed, and cells were lysed with dimethylsulfoxide. Absorbance was measured at 490 nm using a microplate reader (Elx800, BioTek, USA). Relative cell viability was calculated as a percentage of the control group, to which QDs had not been added. 2.5. Annexin V-PE/7-AAD assay

excluding debris. Mitochondrial depolarization was indicated by a decrease in the red/green fluorescence intensity ratio. Confocal micrographs of HEK293 cells stained with JC-1 were acquired with Eclipse C1-si laser scanning confocal microscope system (Nikon). 2.7. Cell cycle analysis Cells were treated with various concentrations of QDs for 48 h. Treated cells were removed by trypsinization and collected by centrifugation at 1500 rpm for 10 min. The cell pellets were resuspended in 70% ethanol and stored at 4 °C for 24 h. After the indicated time, cells were centrifuged and washed twice with PBS buffer. Then we added 50 lL of a 100 lg mL 1 stock of RNase to cell pellets, and stained cell pellets with propidium iodide (PI, 20 lg mL 1 in PBS) for 30 min. Cells were analyzed by flow cytometry (FACSAriaTM III, Becton Dickinson, USA). The forward scatter (FS) and side scatter (SS) were measured to identify single cells. Pulse processing was used to exclude cell doublets from the analysis. PI has a maximum emission of 605 nm so can be measured with a suitable bandpass filter. For analysis, first gate on the single cell population using pulse width vs. pulse area. Then apply this gate to the scatter plot and gate out obvious debris. Combine the gates and apply to the PI histogram plot. 2.8. Reactive oxygen species assay Cells cultured as described above were treated with QDs for 24 h. After incubation with QDs, the culture medium was removed. Then cells were trypsinized, washed twice with PBS buffer. Reactive oxygen species were detected by ROS detection kit (Genmed, USA) according to the manufacturer’s instructions. Cell pellets were added with Genmed binding buffer and then incubated at 37 °C, 5% CO2, for 20 min in the dark. Cells were analyzed on FE detector of flow cytometry (FACSAriaTM III, Becton Dickinson, USA). For statistical significance, at least 10 000 cells were analyzed in each sample.

Cells positive of apoptosis and necrosis were determined by the annexin V-PE/7-AAD assay. The externalization of phosphatidylserine as a marker of early-stage apoptosis was measured by the annexin V protein conjugated to PE, whereas membrane damage due to late-stage apoptosis or necrosis was detected by the binding of 7-AAD to nuclear DNA. Apoptotic cells were measured according to the manufacture’s protocol. Cells cultured as described above were treated with QDs for 24 h. After incubation with QDs, the culture medium was removed. Then cells were trypsinized, washed twice with PBS buffer. 100 lL sample (4  104 cells) was added with 100 lL Annexin V-PE/7-ADD binding buffer and then incubated for 20 min at room temperature in the dark. Cells were analyzed by flow cytometry (FACSAriaTM III, Becton Dickinson, USA). For statistical significance, at least 10 000 cells were analyzed in each sample.

The generation of hydroxyl radicals in the culture medium was determined using a fluorimetric assay (Rajendran et al., 2009). MPA-QDs (5 lM) and disodium terephthalate (500 lM) were incubated with the culture medium. When hydroxyl radicals generate from QDs, terephthalate will be transformed into 2-hydroxyterephthalate, which is highly fluorescent in the presence of sodium hydroxide. The fluorescence emission spectra of 2-hydroxyterephthalate at 428 nm (kex = 300 nm) was monitored on a LS-55 Spectrofluorimeter (Perkin–Elmer, USA).

2.6. Evaluation of mitochondrial membrane potential (Dwm)

2.10. Agarose gel electrophoresis

For quantitative analysis of Dwm, QDs-treated HEK293 cells were also rinsed twice with PBS, trypsinized and then stained with JC-1. 1 mL sample (1  106 cells) was added with 10 lL of 200 lM JC-1 binding buffer and then incubated at 37 °C, 5% CO2, for 20 min in the dark. JC-1 is a lipophilic probe which potential-dependently accumulated in mitochondria and its fluorescence emission shifts from red (590 nm, J-aggregates) to green (525 nm, J-monomers) when mitochondrial membrane potential decreases. Consequently, cell pellets were analyzed on a flow cytometer with 488 nm excitation. The fluorescence of J-aggregates in healthy cells was quantified on PE detector, and green JC-1monomers in apoptotic cells were quantified on FITC detector of flow cytometer (FACSAriaTM III, Becton Dickinson, USA). For statistical significance, at least 10 000 cells were analyzed in each sample. Gated on the cells

CdTe QDs modified with MPA were added to PBS buffer (2 mL), serum-containing DMEM (2 mL) and serum-free DMEM (2 mL), respectively. The final concentrations of QDs were fixed at 100 nmol L 1. The sample were separated on a 0.5% or 0.8% agarose gel electrophoresis. The bands were photographed with Chemi Doc XRS (Bio-Rad Laboratories, USA).

2.9. Fluorimetric hydroxyl radical assay

2.11. Method of high performance liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS) After incubation with QDs (200 nmol L 1) for 24 h, cells were trypsinized, and then dialyze against ultrapure water for 24 h. The amount of tellurium species were determined using high performance liquid chromatography-inductively coupled plasma-

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mass spectrometry (HPLC-ICP-MS). The high performance liquid chromatography (HPLC) module system consisted of an Agilent 1200 Series (Agilent Technologies, Japan). Separation of the tellurium species were performed on a Hamilton PRP-X100 (Hamilton, Reno, NV, USA) anion-exchange column (250 mm  4.1 mm, 10 lm). Mobile phase: 40 mM NH4H2PO4 at pH = 6 with ammonia (Orero Iserte et al., 2004). The inductively coupled plasma-mass spectrometry (ICP-MS) system consisted of an ICPMS Agilent 7700 Series (Agilent Technologies, Japan).

2.12. Laser scanning confocal microscopy Confocal micrographs of cells were acquired with Eclipse C1-si laser scanning confocal microscope system (Nikon, Japan). QDs were added to designated wells, and the cells were incubated for different time periods. Plasma membrane damage was investigated by using propidium iodide (PI, 10 lg mL 1, 10 min). The excitation laser and filter used for PI imaging were HeNe543 nm and LP560, respectively. Nuclei were stained with Hoechst 33 342 (50 lg mL 1, 30 min). Lysosome were stained with Lyso-Tracker Red (75 nM, 30 min, kex 577 nm, kem 590 nm). Before imaging, cells were wash with PBS. No background fluorescence of cells was detected under the settings used.

2.13. Transmission electron microscopy of cell Cells under various experimental conditions were fixed for 30 min at 4 °C using glutaraldehyde at a final concentration of 2.5% in 0.1 M cacodylate buffer, then postfixed with 1% osmium tetroxide and dehydrated. Observations were performed on a JEM-100CX transmission electron microscope (JEOL, Peabody, MA).

2.14. Statistical analysis All the experiments were performed using four independent experiments with different cell preparations. The values are expressed as mean ± SE. Means were compared using ANOVA (paired t-test). Statistical significance was set as P < 0.05.

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3.2. Effect of CdTe QDs on the cell cycle of HEK293 Toxicants can induce a wide collection of cellular responses. Researchers particularly emphasized on the mechanisms for cell death. However, these mechanisms can also be complicatedly linked with the cell cycle, which corresponds to an array of events leading the cells to division and duplication (Mahmoudi et al., 2011). As shown in Figs. S8 and S9, we examined whether MPACdTe QDs blocked the cell cycle of HEK293 and HeLa cells using flow cytometry. The cell cycle distribution of HEK293 and HeLa cells after MPA-CdTe QDs treatment for 48 h are presented in Tables S1 and S2. The cell cycle profile remained similar among the treatments. No significant cell cycle arrest and no appreciable fraction of cells with sub-G1 DNA content were observed and detected. These results indicate that MPA-CdTe QDs did not induce apoptotic cell death. 3.3. CdTe QDs lead to necrotic cell death of HEK293 To further confirm the mechanism for cell death, we detected the apoptosis or necrosis in HEK293 cells treated with different concentrations of CdTe QDs for 24 h by flow cytometry, using an annexin V-PE/7-AAD apoptosis detection kit. After 24 h incubation with QDs, the percentages of annexin V-positive/7-AAD-positive HEK293 cells (Fig. 1 and Table S3) increased gradually with the increasing of CdTe QDs concentration (4.5% at control, 8.8% at 50 nM, 18.1% at 100 nM and 29.5% at 200 nM). When cell membrane loses its integrity, the cell becomes both annexin V and 7AAD positive, suggesting cell necrosis. These results clearly indicate that CdTe QDs could lead to necrotic cell death for HEK293 cells. Propidium iodide (PI), a red fluorescent probe, cannot enter into cells when the plasma membrane is intact. Cell necrosis leads to the loss of membrane integrity, therefore cell necrosis can be examined via the level of PI uptake. Figs. S10 and S11 show confocal micrographs of HEK293 and HeLa cells stained with PI after QDs treatment for 48 h. A significant increase of cellular PI uptake was observed after QDs treatment using laser scanning confocal microscopy. This is in accordance with the results obtained by annexin V-PE/7-AAD apoptosis detection. 3.4. Effects of QDs on mitochondrial membrane potential (Dwm)

3. Results 3.1. Cytotoxic effect of CdTe QDs increased in a dose- and timedependent manner We firstly examined the cytotoxicity of MPA-CdTe QDs directly synthesized in aqueous phase targeted to cell lines including HEK293 and HeLa. The cytotoxicity of QDs was evaluated by the effect on the cellular metabolic viability through MTT assay and morphological observation of cells with bright-field microscopy. As shown in Fig. S6(a) and (b), a significant decrease in the mitochondrial metabolic activity of HEK293 and HeLa cells treated with MPA-CdTe QDs was observed. Cell metabolic viability dropped dose-dependently with the increase of QDs concentration. The TC50 values after 48 h of exposure to HEK293 and HeLa cells were found at 109.4 and 65.29 nmol L 1, respectively. These results indicate that MPA-CdTe QDs possess higher toxic effect to HEK293. In addition, HEK293 cells were treated with 100 nM MPA-CdTe QDs for different time. As shown in Fig. S6(c), the cell viability of HEK293 decreased time-dependently with the addition of QDs. MTT assays of viability can be confirmed via morphological observation of cells through bright-field microscopy. As shown in Fig. S7, cells exposed to QDs rounded up and showed indistinct intercellular boundary.

Mitochondrial permeability transition (MPT) is an important step in the induction of cellular apoptosis and necrosis (Yang et al., 2007). Fig. 2 shows the effect of MPA-CdTe QDs on mitochondrial membrane potential determined by flow cytometry. Collapse of the mitochondrial DWm is indicated by an increase in the number of cells falling into P2 gate corresponding to a loss of red fluorescence, indicative of the onset of DWm depolarizing event. Percentage of HEK293 cells in P2 gate determined by JC-1 staining are listed in Table S4. MPA-CdTe QDs caused a pronounced reduction of Dwm, as the percentage of unhealthy HEK293 cells with low Dwm were remarkably increased from 6.9% to 43.5% in the presence of 200 nM CdTe QDs. These results were also verified by the confocal micrographs of HEK293 cells stained with JC-1 (Fig. S14). Healthy cells exhibited orange-red stained mitochondria. When incubated with 50 nmol L 1 MPA-CdTe QDs for 24 h, HEK293 cells contained less orange-red fluorescing J-aggregate and appeared mostly green. 3.5. Ultrastructural changes in HEK293 cells In order to study the ultrastructural changes in cells treated with QDs, TEM analyses of the HEK293 cells treated with 100 nmol L 1 of MPA-CdTe QDs were performed. Untreated cells

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Fig. 1. Detection of MPA-CdTe QDs induced apoptosis with Annexin V-PE and 7-AAD staining. HEK293 cells were incubated with different doses of MPA-CdTe QDs for 24 h.

showed no abnormalities (Fig. S15(a) and (b)), whereas QDs treated cells showed a series of morphological change (Fig. S15(c)– (k)). Nuclear condensation with DNA fragmentation is a mark of cell apoptosis, whereas, the nuclei of necrotic cells were swollen (Fig. S15(f) and (k)). We have previously shown that QDs induced mitochondrial damage in HEK293 cells using JC-1 staining. Ultrastructural resolution confirmed that QDs induced swelling of mitochondria (Fig. S15 (d), (h) and (j)). In addition, we observed other ultrastructural changes, such as condensation and margination of cytoplasm (Fig. S15(f) and (k)), nuclear membrane folding (Fig. S15(j)), endoplasmic reticulum expansion (Fig. S15(h)), ribosomal aggregation (Fig. S15(d), (h) and (j)), and a marked increase in vesica and lysosome number (Fig. S15(c), (e) and (i)). 3.6. The toxicity mechanism of MPA-CdTe QDs It is reported that tellurium is very toxic and teratogenic. The doubly negatively charged tellurium ion is readily oxidized, and therefore tellurium may exist in other chemical forms. Anion exchange LC-ICP-MS coupling has become one of the most widely used techniques for tellurium speciation studies and the detection limit achieved were at sub-lg
L 1 levels (Orero Iserte et al., 2004). To measure the total tellurium concentration leaching from the surface of QDs during cell culture, we added different amount of CdTe QDs in culture media. The concentrations of QDs ranged from 25 to 100 nmol L 1. After 48 h incubation, the mixture of DMEM and QDs was collected and dialyzed against ultrapure water for

24 h. The results of LC-ICP-MS experiments showed the total tellurium concentration is quite low and out of the limit of detection. Considering that the precursor Cd/Te molar ratio is 5 in the synthesis of MPA-CdTe QDs, thus the elements of QDs surface are mainly cadmium and sulfur. Therefore, the inhibition effect of tellurium on the cellular metabolic viability was neglected in the successive research. In order to further study whether the reduction of metabolic activity of HEK293 cells is caused solely by the release of cadmium ions or not, we carried out the similar experiment. The HEK293 cell were incubated with QDs solution, CdCl2 solution, MPA solution and the mixture of CdCl2 and MPA, respectively. In this experiments, the concentration of MPA-QDs is fixed at 100 nmol L 1. The results of ICP-AES showed that the 100 nmol L 1 MPA-CdTe QDs solution used in this paper averagely contain 8 lmol L 1 Cd2+, thus the concentration of CdCl2 is selected as 8 lmol L 1. And the molar ratio of MPA and Cd is 1.7 in the synthesis of MPA-CdTe QDs, so we fixed the concentration of MPA at 13.6 lmol L 1. As shown in Fig. S16, the MPA-QDs significantly decreased the metabolic activity of HEK293 cells, while cadmium ions induced a small reduction in metabolic activity. Additionally, we examined the effect of surface ligands on the cytotoxicity of QDs. To this end, we prepared four types of CdTe quantum dots with nearly identical maximum emission wavelength (520 nm) but capped with different ligands. The effects of ligands on the cytotoxicity of quantum dots are presented in Fig. S17. At a concentration of 50 nmol L 1, the toxicity order of

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Fig. 2. Effects of MPA-CdTe QDs on mitochondrial membrane potential. HEK293 cells were incubated with different doses of MPA-CdTe QDs for 24 h.

QDs is MPA > GSH > NAC > dBSA. The QDs modified with 3Mercaptopropionic acid (MPA) had higher toxicity than the others, whereas the presence of dBSA greatly improved the biocompatibility of QDs. However, when the concentration of CdTe QDs reaches 100 nmol L 1, we can observed that dBSA-CdTe QDs have also exhibited high cytotoxicity and significantly reduced the metabolic activity of HEK293 cells, although the toxicity order of QDs remains MPA > GSH > NAC > BSA. Consequently, we speculate that ligands modified on the surface of CdTe QDs cannot effectively alleviate the cytotoxicity of QDs at the higher concentration of QDs. Owing to the large surface area to volume ratio, QDs have strong tendency to interact with biomacromolecules in culture medium. Once biomolecules are adsorbed onto QDs surface, the original surface ligands of QDs have no perceptible effect on the cytotoxixity of QDs. 3.7. Reactive oxygen species (ROS) induced by MPA-CdTe QDs The generation of reactive oxygen species is another primary mechanisms of nanotoxicity. We detected the intracellular ROS levels by the use of dihydro-ethidium (DHE), a fluorescent probe dye that exhibits increased red fluorescence intensity when oxidized by ROS. As shown in Fig. 3(a), a dose-dependent increase of ROS level was observed in cells after 24 h of QDs exposure. In addition, we determined the generation of hydroxyl radicals in the culture medium using a fluorimetric assay (Rajendran et al., 2009). MPA-QDs (5 lM) and disodium terephthalate (500 lM) were incubated with the serum-containing medium in the absence of cells. When hydroxyl radicals generate from QDs, terephthalate will be transformed into 2-hydroxyterephthalate, which is highly fluorescent in the presence of sodium hydroxide. We monitored the fluorescence emission spectroscopy of 2-hydroxyterephthalate at 428 nm (kex = 300 nm) for 24 h. As shown in Fig. 3(b), the

negative control (without QDs) did not exhibit significant fluorescence emission at 428 nm, while no obvious increase in fluorescence intensity at 428 nm was observed in the culture medium treated with QDs. In contrast, the positive control treated with H2O2 but without QDs is remarkable 4-fold increase in the fluorescence intensity at 428 nm. Since we did not intentionally expose cells to the light, we suggest that ROS do not directly generate from QDs in the culture medium. Long et al. demonstrated ROS generation is an initial cellular response to nanoparticle internalization (Long et al., 2005). As a result, increased ROS production has been associated with the internalization of QDs with cells.

3.8. The interaction of CdTe QDs with serum proteins Understanding of the interaction of QDs with culture medium can contributed to the explanation of cytotoxicity mechanism of QDs. Therefore, the photoluminescence spectra of MPA-CdTe QDs in serum-containing DMEM (10% FBS) were monitored for 48 h. As shown in Fig. S18(a), the culture medium is non-fluorescent in the spectral range, whereas the emission peak intensity of QDs decreased slightly during the first 2 h. However, the intensities of QDs emission gradually decrease as the incubation time further increase while the emission peak wavelength hardly change (Fig. S18(b)). Additionally, we investigated the effect of culture medium on absorption spectra of quantum dots. A certain amount of QDs was added in 3 mL serum-containing DMEM (10% FBS). When DMEM (10% FBS) was used as a reference solution, absorption spectra of quantum dots were monitored for 120 min. As shown in Fig. S19, the first electronic transition absorption peak of QDs graduate become ill-defined as the incubation time further increase. Simultaneously, the negative peak was observed near

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Fig. 3. (a) Reactive oxygen species (ROS) formation in HEK293 cells as a function of the exposure to 0, 25, 50, 100, 200 nmol L 1 MPA-CdTe QDs for 24 h. (b) Fluorescence spectra of 2-hydroxyterephthalate anion generation in the culture medium untreated with QDs, treated with QDs, treated with H2O2 for 24 h, respectively. (c) The interaction of MPA-QDs with culture medium determined by gel electrophoresis. [QDs] = 100 nmol L 1.

560 nm. Taken together, these results demonstrated some biomolecules in the culture medium probably adsorb on the QDs surface. Furthermore, in order to clarify the interaction mechanism of different culture medium component with QDs, we investigated the interaction of serum-containing or serum-free DMEM with QDs using agarose gel electrophoresis. A certain amount of QDs were added in 2 mL DMEM containing 10% FBS and serum-free DMEM, respectively. The final concentrations of QDs are 100 nmol
L 1. The sample were separated on a 0.5% or 0.8% agarose gel electrophoresis and the bands were photographed (Fig. 3(c)). In Fig. 3(c), an agarose gel is depicted, QDs have been loaded in the left lane. In the middle lane, the mixture of QDs and serum-containing DMEM was loaded, while the mixture of QDs and serumfree DMEM was loaded in the right lane. Significantly, it was observed that the migration of QDs was not much different between the left and right lane, whereas QDs added in the serum-containing DMEM travel slower than others (Fig. 3(c)). For agarose gel electrophoresis, biomolecules are separated by applying an electric field to travel the charged molecules through an agarose matrix, and the biomolecules are separated by size and charge in the matrix. Due to large surface/volume ratios of quantum dots, they are extremely reactive and possess high surface energy. When dispersed in culture medium, quantum dots are prone to reduce their large surface energy by adsorption with serum proteins and other biomacromolecules. The adsorption of serum proteins on the QDs surface will change the size and surface charge of QDs, resulting in the change of electrophoresis rate. When added in culture medium, QDs mainly interact with serum proteins. Fluorescence is the process of photon emission as a result of the return of an electron in a higher energy orbital back to a lower orbital. A variety of molecular interactions can result in fluorescence quenching, including excited-state reactions, energy transfer, ground-state complex formation, and collisional quenching

(Lai et al., 2012). Therefore, we investigated the interaction of QDs with fatal bovine serum using fluorescence spectroscopy. 3 mL 10% FBS was titrated manually by successive additions of QDs. The final concentrations of QDs varied from 0 to 10 lmol L 1 at increments of 1 lmol L 1. The fluorescence spectroscopy were monitored. As shown in Fig. S20, a progressive decrease in the fluorescence intensity of fatal bovine serum was caused by quenching, accompanied by a red shift of maximum emission wavelength from 345.5 to 358.0 nm. According to the previous reports, the red shift can reasonably be attributed to an increased polarity of the region surrounding the tryptophan site (Lai et al., 2012). Proteins in FBS may be adsorbed onto negatively charged quantum dots to form a ‘‘protein corona’’ (Cedervall et al., 2007). The adsorption of protein on the surface of QDs will induce a slight unfolding of the protein polypeptides and this resulted in a conformational change of the protein that will increase the exposure of tryptophan site that had initial been buried to more polar environment. The conformational changes of protein may due to the surface curvature of quantum dots when protein is adsorbed. At the same time, we observed a red shift of maximum emission wavelength of QDs from 529.5 to 533.5 nm, also indicating that protein adsorbed on the surface of QDs. The interaction between fetal bovine serum and QDs may change the surface properties of QDs, including the change of zeta potential and size, which may influence the interaction of QDs and cells. 3.9. The cellular uptake, location, and cytotoxicity of CdTe QDs A classic clathrin-mediated endocytosis inhibitor, chlorpromazine, was added to examine whether the uptake of QDs can be inhibited and consequently reduces cytotoxicity of QDs. Firstly, we determined the effect of chlorpromazine on the cell viability by MTT assays. As shown in Fig. S21, HEK293 cells viability declined at the concentration of chlorpromazine higher than

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1.2

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the co-localization experiment. As shown in Fig. 5(b), QDs mainly adsorbed on the cell membrane of HEK293.

Cell viability / %

1.0

4. Discussion

0.8 0.6 0.4 0.2 0.0

[QDs] 0 [Chlorpromazine] 0

50nM 100nM 200nM 2μΜ 4μΜ 8μΜ 2μΜ 4μΜ 8μΜ 2μΜ 4μΜ 8μΜ

Fig. 4. Metabolic activity of HEK293 cells treated with QDs (50, 100, 200 nmol L 1) in the absence and presence of chlorpromazine (2, 4, 8 lmol L 1) for 48 h. Error bars represent standard errors of the mean values.

8 lmol L 1. Therefore, the highest concentration of chlorpromazine is fixed at 8 lmol L 1 in the following experiments. The metabolic activity of cells treated with different concentrations of QDs (50, 100 and 200 nmol L 1, respectively) in the absence and presence of chlorpromazine are present in Fig. 4. It was observed from Fig. 4 that the addition of chlorpromazine reduce the cytotoxicity of QDs. In this work, we observed the localization of QDs in HEK293 cells cultured in serum-containing DMEM using confocal fluorescence microscopy. As shown in Fig. 5(a), co-localization of the CdTe QDs (green) with Lyso-Tracker (red) exhibited a significant level of lysosomal sequestration. The surface properties of QDs is one of the most essential factors related to their cellular uptake. In order to determine the effect of protein adsorption on the cellular uptake of QDs, we cultured HEK293 cells in opti-MEM (reduced serum medium) and duplicate

This work presents the toxicity of different ligand-coated CdTe QDs to HEK293 and HeLa cells. When treated with CdTe QDs, the cell becomes both annexin V and 7-AAD positive, suggesting cell necrosis. Mitochondrial permeability transition (MPT) is an important step in the induction of cellular apoptosis and necrosis (Yang et al., 2007). Fig. 2 indicated that MPA-CdTe QDs caused a pronounced reduction of Dwm. Some previous reported researches attributed the toxic effect of quantum dots to the release of Cd2+ (Derfus et al., 2004), the generation of ROS (Chibli et al., 2011), the detachment of ligands (Hoshino et al., 2004), etc. Among these factors, the first to be considered is the inherently toxic elements of QDs (e.g., cadmium, tellurium) leaching from the surface of QDs. The release of Cd2+ is always the center of discussion related to the cytotoxicity of cadmium containing QDs. Derfus et al. proposed that surface oxidation led to the formation of reduced Cd on the QDs surface, release of free cadmium ions, led to cell death. Surface coating could significantly reduce but not eliminate cytotoxicity (Derfus et al., 2004). Moreover, Su et al. determined the concentration of intracellular cadmium ions and total cadmium for cells treated with QDs. They found that the cytotoxicity of CdTe QDs is greater than CdCl2 solution of intracellular cadmium ions concentration. However, there was a good correlation between the cytotoxicity and the total intracellular cadmium content of QDs (Su et al., 2010). Chen et al. have also confirmed that the growth rates of HEK293 cells treated with 5 lM and 10 lM CdCl2 were not significantly changed as compared to untreated cells (Chen et al., 2012). In another report, Su has also found that no obvious morphological change of HEK293 cells was observed in the presence of 20 lM CdCl2 (Su et al., 2010). Simultaneously, our results shown in Fig. S16 indicated the toxic effect is not prominent when HEK293 cells were exposured to MPA or the mixture of CdCl2 and MPA. Therefore, according to these previous reports and our results,

Fig. 5. (a) QDs colocalization with Lyso-Tracker Red. HEK293 cells were incubated with QDs in serum-containing DMEM (10% FBS) for 8 h. (b) QDs colocalization with LysoTracker Red. HEK293 cells were incubated with QDs in Opti-MEM for 8 h.

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we speculate the release of cadmium ions cannot be the main reason for the cytotoxicity of cadmium-based quantum dots. Nanomaterials have a greater reactive surface area than bulk materials, and thus produce greater numbers of reactive oxygen species. ROS may result in oxidative stress, inflammation, and consequent damage to DNA, lipid peroxidation, oxidations of amino acids in proteins, etc. Reactive oxygen species (ROS) includes both oxygen radicals, such as superoxide, hydroxyl, peroxyl, and hydroperoxyl radicals, and certain nonradical oxidizing agents, such as hydrogen peroxide, hypochlorous acid, and ozone. Nanomaterial can transfer energy to nearby oxygen molecules in the culture medium to induce the generation of reactive oxygen species in the presence of light. Niemeyer et al. reported the generation of free radical species from quantum dots upon UV irradiation in aqueous solution (Ipe et al., 2005). They demonstrated the generation of radical species depends on the QDs material. CdS QDs apparently generated both hydroxyl and superoxide radicals, while CdSe QDs solely generated hydroxyl radicals. On the contrary, CdSe/ZnS QDs did not produce free radicals. Since the redox potential of O2 ? O2 is 0.15 eV, the reduction power of the conduction band potential of CdTe is not enough to reduce O2. The oxidation potential of hydroxide to OH radicals is 1.9 eV, therefore the VB (valence band) edges of CdTe is insufficient to oxidize H2O or hydroxide ions to
OH radicals (Ipe et al., 2005). As shown in Fig. 3(b), no obvious increase in fluorescence intensity at 428 nm was observed in the culture medium treated with QDs. Since we did not intentionally expose cells to the light, we suggest that ROS do not directly generate from QDs in the culture medium. Long et al. demonstrated ROS generation is an initial cellular response to nanoparticle internalization (Long et al., 2005). As a result, increased ROS production has been associated with the internalization of QDs with cells. A thorough understanding of the uptake and trafficking processes of QDs is critical in determining the mechanism of QDs induced cytotoxicity. Cell membrane is generally to serve as a permeability barrier, which prevents the entry of toxic compounds but allows the influx of nutrient molecules (Lai et al., 2013). Small molecules move across the cell membrane through directly diffuse driven by their concentration gradients, integral membrane protein pumps or ion channels, whereas nanoscale biomolecules are conventionally pass through the cell membrane by endocytosis (Zhao et al., 2011). It is reported that the endocytotic processes of nanomaterials by cells mainly include pinocytosis, phagocytosis, caveolae-dependent endocytosis, and clathrin-mediated endocytosis (Zhao et al., 2011). It was observed from Fig. 4 that the addition of chlorpromazine reduce the cytotoxicity of QDs in a dose-dependent manner. Consequently, the clathrin-mediated endocytosis might be the important cellular uptake pathway for CdTe QDs by HEK293 cells, resulting in the cytotoxicity of QDs.

After QDs were internalized by cells, the next important question is their intracellular location, which is directly related to the cytotoxicity of the internalized QDs. In the clathrin-mediated endocytic processes, QDs will be enclosed in small endocytic vesicles. After entering cells, these endocytic vesicles will fuse with early endosomes. The translocation of QDs in cells is usually via endosomal or lysosomal vesicles. Lysosomal are spherical vesicles containing more than fifty different enzymes which are all active at an acidic environment of about pH 5. Therefore, lysosomal can digest the internalized QDs. The rapid degradation of QDs will lead to the release of toxic substance into the cytosol, and then induce cell necrosis. Taken together, when QDs added in the culture medium, serum proteins in the culture medium will be adsorbed on the surface of QDs. This adsorption of serum protein will change the surface properties and size, and then mediate the cellular uptake of QDs via the clathrin-mediated endocytic pathway. After entering in cells, the translocation of QDs in cells is usually via endosomal or lysosomal vesicles. The rapid degradation of QDs in lysosome induce cell necrosis (Fig. 6). 5. Conclusion This work presents the toxicity of different ligand-coated CdTe QDs to HEK293 and HeLa cells. The results showed that the cytotoxicity of QDs increased in a dose- and time-dependent manner. The results of cell cycle analysis and annexin V-PE/7-AAD assay indicated that CdTe QDs could lead to necrotic cell death in HEK293 cells. Mitochondrial swelling and collapse of the membrane potential confirmed that QDs induced MPT. Moreover, QDs induced MPT through the elevation of ROS. The fluorimetric assay and theoretical analysis demonstrated ROS production has been associated with the internalization of QDs with cells. When QDs added in the culture medium, serum proteins in the culture medium will be adsorbed on the surface of QDs. This adsorption of serum protein will mediate the cellular uptake of QDs via the clathrin-mediated endocytic pathway. After entering into cells, the translocation of QDs in cells is usually via endosomal or lysosomal vesicles. The rapid degradation of QDs in lysosome and the lysosomal destabilization induce cell necrosis. Acknowledgements The authors gratefully acknowledge the financial support from Chinese 973 Program (Grant No. 2011CB933600), National Science Fund for Distinguished Young Scholars of China (Grant No. 21225313), National Natural Science Foundation of China (Grant Nos. 21403017, 21473125, 21303126), Educational Commission of Hubei Province of China (Grant Nos. Q21473125, Q20141302), Large-scale Instrument And Equipment Sharing Foundation of Wuhan University and Fundamental Research Funds for the Central Universities (Grant No. 2042014kf0287). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemosphere. 2015.04.044. References

Fig. 6. The cytotoxicity mechanism of CdTe QDs on HEK293 cells.

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