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Toxicology Letters 221 (2013) 118–127

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Single-walled carbon nanotubes and graphene oxides induce autophagosome accumulation and lysosome impairment in primarily cultured murine peritoneal macrophages Bin Wan ∗,1 , Zi-Xia Wang 1 , Qi-Yan Lv, Ping-Xuan Dong, Li-Xia Zhao, Yu Yang, Liang-Hong Guo ∗ State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 10085, China

h i g h l i g h t s • • • • •

Two carbon nanomaterials were used to study the cytotoxicity in macrophages. Both carbon nanomaterials induced autophagosome accumulation. Both carbon nanomaterials blocked autophagy flux. Carbon nanomaterials accumulated in lysosomes and caused lysosome destabilization. Graphene oxides were more potent than carbon nanotubes in inducing cell responses.

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Article history: Received 18 April 2013 Received in revised form 30 May 2013 Accepted 5 June 2013 Available online xxx Keywords: Carbon nanomaterials Macrophage Autophagy Autophagosome accumulation Lysosome impairment

a b s t r a c t The wide application of carbon nanomaterials in various fields urges in-depth understanding of the toxic effects and underlying mechanisms of these materials on biological systems. Cell autophagy was recently recognized as an important lysosome-based pathway of cell death, and autophagosome accumulation has been found to be associated with the exposure of various nanoparticles, but the underlying mechanisms are still uncertain due to the fact that autophagosome accumulation can result from autophagy induction and/or autophagy blockade. In this study, we first evaluated the toxicity of acid-functionalized single-walled carbon nanotubes and graphene oxides, and found that both carbon nanomaterials induced adverse effects in murine peritoneal macrophages, and GOs were more potent than AF-SWCNTs. Both carbon nanomaterials induced autophagosome accumulation and the conversion of LC3-I to LC3-II. However, degradation of the autophagic substrate p62 protein was also inhibited by both nanomaterials. Further analyses on lysosomes revealed that both carbon nanomaterials accumulated in macrophage lysosomes, leading to lysosome membrane destabilization, which indicates reduced autophagic degradation. The effects of AF-SWCNTs and GOs on cell autophagy revealed by this study may shed light on the potential toxic mechanism and suggest caution on their utilization. © 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Carbon nanomaterials are an important family of nanomaterials because of their unique physicochemical properties that represent wide range of potential applications (Ball, 2001). Among them, two-dimensional graphene and its derivatives (e.g. graphene oxide,

∗ Corresponding authors at: State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-environmental Sciences, The Chinese Academy of Science, 18 Shuangqing Road, P.O. Box 2871, Beijing 100085, China. Tel.: +86 10 6284 9338; fax: +86 10 6284 9685. E-mail addresses: [email protected] (B. Wan), [email protected] (L.-H. Guo). 1 These authors contributed equally to this work. 0378-4274/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2013.06.208

reduced graphene oxide), as a rising star of carbon nanomaterials, have been extensively investigated and used for supporting the growth and adhesion of live cells (Chen et al., 2008; Agarwal et al., 2010), cell labeling and imaging (Li et al., 2012; Caravan et al., 1999), and gene delivery (Chen et al., 2011; Kim et al., 2011). The onedimensional carbon nanotube (CNT) is one of the most prominent nanomaterials (Bianco et al., 2005). Functionalized single-walled carbon nanotubes (f-SWCNTs) are valuable nanomaterials in many industries, especially in biomedical applications (Chen et al., 2008; Bianco et al., 2005). The wide application of the carbon nanomaterials urged indepth investigation on their potential toxic effects on biological systems and the underlying mechanisms. During the last decade, many efforts have been concentrated on understanding the side

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effects of CNTs and GOs both in vivo and in vitro. For example, CNTs were found to suppress systemic immune function (Mitchell et al., 2009) and impair phagocytic function of monocytes/macrophages (Jia et al., 2005; Dong et al., 2012a). At the cellular level, in vitro studies have found that CNTs were able to induce lower cell viability as well as altered cell adhesion and cell cycle regulation (Magrez et al., 2006; Cui et al., 2005). In addition, several lines of evidences found that GOs not only enhanced the attachment and proliferation of bacteria and mammalian cells (Ruiz et al., 2011), but also promoted stem cell differentiation (Lee et al., 2011). Studies also showed that graphene and GOs damaged cell membrane physically (Hu et al., 2011), changed mitochondrial membrane potential, increased intracellular reactive oxygen species and activated apoptosis (Li et al., 2012; Duch et al., 2011). In general, the mode of cell death is dependent on the cell type, energy metabolism and level, signaling pathway, stimulus, and environment. Thus there exist various intermediate forms of cell death displaying both apoptotic and necrotic characteristics (Ziegler and Groscurth, 2004). Previous studies of ours and others have showed that cell exposed to carbon nanomaterials might end in an apoptosis-like death without activation of caspases, reflecting an alternative pathway (Dong et al., 2012a,b; Shvedova et al., 2005), but the underlying mechanisms were not well understood. Recently, a lysosome-based degradation process named cell autophagy was proposed as an important pathway of cell death (Stern et al., 2012). Autophagy involves cell degradation of unnecessary or dysfunctional cellular components through the lysosomal machinery. During this process, targeted cytoplasmic constituents are isolated from the rest of the cell within the double-membraned autophagosome, which are then fused with lysosomes and degraded or recycled. Cellular autophagic activity is usually low under basal condition, but can be markedly upregulated by numerous stimuli such as nutrient starvation, innate immune signals, chemical reagents, and infections. Autophagy has been seen as an adaptive response to survival, whereas unstrained autophagy appears to promote cell death and morbidity (Mizushima et al., 2010; Patel et al., 2012). Autophagy can be monitored by quantifying the number of autophagosomes, which form during the initiation of autophagy and are degraded at the end. Microtubule-associated light chain 3 (LC3) is a marker of autophagosomes, and nascent LC3 is processed at its C terminus by Atg4 and becomes LC3-I, which subsequently conjugates with phosphatidylethanolamine (PE) to become LC3-II; LC3-II then translocates to the autophagosome membrane (Mizushima et al., 2010). Therefore, in addition to direct observation of autophagosomes through electron microscopy, fluorescence microscopic analysis on the formation of LC3 punctas and biochemical analysis on the conversion of LC3-I to LC3-II are also widely used for monitoring autophagosome formation (Ma et al., 2011). In deed, nanomaterials have recently been recognized as a novel class of autophagy activators probably due to their physical similarity with particular viruses, bacteria, and parasites (Zabirnyk et al., 2007). A variety of nanomaterials, including metal oxides (Kenzaoui et al., 2012; Hussain and Garantziotis, 2013; Sun et al., 2012), carbon nanomaterials (Chen et al., 2012; Liu et al., 2011), gold nanomaterials (Li et al., 2010), were demonstrated to induce autophagosome accumulation. Nevertheless, some of above studies neglected the fact that the number of autophagosomes is a function of the rate of its generation and the rate of its degradation at any specific time point. Blockade of autophagy flux and autophagy induction can both lead to autophagosome accumulation. The possibility of autophagy blockade was often not investigated, and the mechanism in many cases is uncertain (Mizushima et al., 2010). Specifically, carboxylic acid functionalized SWCNTs and GOs were shown to induce autophagosome accumulation and cytotoxicity in human lung cells and RAW264.7 macrophages, respectively (Chen

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et al., 2012; Liu et al., 2011). However, both studies focused merely on the induction of autophagy, while ignoring possible suppression of the steps in the autophagy pathway downstream of autophagosome formation, which has been recently suggested as a more likely mechanism of nanomaterial-induced toxicity (Stern et al., 2012). In this study, we used primarily cultured murine peritoneal macrophages as an ex vivo model to investigate whether both carbon nanomaterials were able to induce autophagosome accumulation and autophagy blockade. The results might shed light on the underlying mechanisms of carbon nanomaterial-induced cytotoxicity. 2. Materials and methods 2.1. Preparation of graphene oxides (GOs) and acid functionalized single-wall carbon nanotubes (AF-SWCNTs) GO was prepared from purified graphite by using the modified Hummers method (Hummers and Offeman, 1958; Zhang et al., 2011). First, graphite powder (1 g), with NaNO3 (1 g), was added to concentrated H2 SO4 (23 mL), and the mixture was stirred for 30 min in an ice bath, followed by gradual addition of KMnO4 (3 g over 40 min). A homogeneous liquid suspension was obtained after sonication for 5 h at 40 kHz to exfoliate the graphene oxide, after which deionised water was added gradually (46 mL over 15 min) and further stirred for 10 min. Then, deionised water (140 mL) and aqueous H2 O2 (30%, 10 mL) were added to the reaction mixture, and unexfoliated graphene oxide was removed by centrifugation (1500 rpm, 10 min). Finally, the sediment was washed 5 times by suspending in HCl solution (5%), followed by washing twice with deionised water and then dehydrated for further use. The resultant powder can be readily resuspended in aqueous solution by sonication, and a stock solution of 1 mg/mL was prepared in deionised water. SWCNT (CNT purity > 95%, SWCNT purity > 90%, ash < 1.5 wt%) synthesized by chemical vapor deposition (CVD) method were originally obtained from Chengdu Organic Chemicals Co. (Sichuan, China). The detailed information can be found on the company website: http://www.timesnano.com/. Acid-functionalisation of SWCNT was performed according to the procedure described previously (Kam et al., 2004; Dong et al., 2012b). In detail, 10 mg of SWCNT were suspended in 40 mL of a 3:1 mixture of concentrated H2 SO4 /HNO3 in a 100 mL test tube and sonicated in a water bath (KQ-250DB, 40 kHz) for 24 h at 40–50 ◦ C. The resultant suspension was then diluted with 200 mL deionised water and filtered through a membrane (pore size 0.22 ␮m), followed by wash with 50 mL deionised water on the membrane. The acid-functionalised nanotubes were re-suspended in deionised water at a concentration of 1 mg/mL with brief sonication (KQ-250DB, 40 kHz, 10 s). The AF-SWCNT suspension was black, well dispersed and had a neutral pH. 2.2. Characterization of AF-SWCNT and GO AFM measurements were performed using a Nanoscope IIIa atomic force microscope (Veeco, USA). After the Graphene sheets were dispersed into water, a few drops of the solution were pipetted on mica substrates. Next, the substrates were air-dried and placed directly under the AFM tip for morphological analysis. For transmission electron microscopy (TEM) characterization, AF-SWCNTs were diluted to 0.01 mg/mL and precipitated onto a copper net and dried for imaging with a Hitachi H-7500 TEM (Tokyo, Japan). A Zetasizer Nano (Malvern Instruments, Malvern, UK) was used for measuring zeta potential (ZP) and hydrodynamic diameters of AFSWCNT and GO suspension. The samples were prepared at a concentration of 10 ␮g/mL in water with neutral pH by the same dispersion method mentioned above. The measurements were repeated three times. In addition, the infrared spectra of AF-SWCNTs and GOs were collected by using a FT-IR spectrometer (JASCO, Inc., Easton, MD, USA), Raman characterization of AF-SWCNTs and GOs were performed on an in Via Raman spectroscopy (Renishaw Plc., Gloucestershire, UK). An Agilent 7500 inductively coupled plasma mass spectrometer (ICP-MS) (Santa Clara, CA, USA) was used to measure the metal content of AF-SWCNTs and GOs, including iron, cobalt, nickel, and manganese ions. 2.3. Cell culture and treatment Mouse peritoneal macrophages were collected and pooled from two to three Kunming female mice injected intra-peritoneally with thioglycollate (TG) broth (3 wt%/vol; 1 mL/mouse; Difco Laboratories, Livonia, MI, USA) 3 days before cell collection to elicit the macrophages into the peritoneal cavity. Cells were plated onto Corning flat-bottomed 6- and 96-well tissue culture plates at 0.5 × 106 to 1 × 106 cells/well and 3 × 105 to 5 × 105 cells/well, respectively, and were incubated at 37 ◦ C, 5% CO2 /95% air, and 95% humidity for 4 h to allow the macrophages to adhere to the surfaces. The surfaces were washed twice with PBS to remove all non-adherent cells, and the macrophage layer was cultured overnight in complete RPMI-1640 (c-RPMI) medium consisting of RPMI-1640 and 10% heat deactivated fetal bovine serum (FBS) supplemented with 20 mM l-glutamine and 100 Uml−1 penicillin/streptomycin. All ingredients for the media were purchased from Hyclone Inc. (Waltham, MA, USA).

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The resulting macrophage purity was 95%, determined by CD11b staining analysis. The macrophages were then exposed to various concentrations of AF-SWCNTs and GOs. The exposure solutions were prepared by diluting stock solutions in cell culture media (the proportion was kept 0.05) (Fig. S1. 2.6. Transient transfection Macrophages were seeded in an 8-well Lab-Tek coverglass the day before being transfected with GFP-LC3 plasmid using Attractene Transfection Reagent (QIAGEN, Venlo, Netherlands). The plasmid DNA and transfection reagents were mixed with the cell culture medium without FBS and incubated for 15 min at room temperature, according to the protocol provided by the manufacturer. Then, the mixture was dropped onto the coverglass and the cells were incubated with c-PRMI-1640 medium. Twenty four hours later, the transfected cells were treated with or without nanomaterials for another 24 h, and the fluorescence images were digitally acquired on a Lecia TCS SP5 confocal laser-scanning microscope (Mannheim, Germany). 2.7. Western blot Cells were lysed with RIPA lysis buffer after treatment, and samples were separated by SDS-PAGE with 15% and 10% separation gel for LC3 and p62 protein detection, respectively. The samples were transferred to PVDF membrane (0.45 ␮m), and the membranes were then blocked with 5% non-fat milk and washed in TBST (containing 0.1% Tween 20). The membranes were incubated with primary antibodies (1:1000 dilution) specific for LC3, P62 and ␤-actin (Cell Signaling Technology, MA, USA) at 4 ◦ C overnight, and washed in TBST again. Then the membranes were incubated with corresponding HRP-conjugated secondary antibody (1:5000 dilutions) for 1 h at room temperature, and washed with TBST. The last step was X-ray film exposure.

set as the quality control. Macrophages were cultured on Thermo Lab-Tek chambered borosilicate coverglass system (Thermo, Rochester, USA) and treated with 10 ␮g/mL AF-SWCNTs and GOs for 24 h. After exposure and wash with PBS, cells were incubated with the two fluorescent probes (Lyso-Tracker Red, 75 nM; Hochest 33342, 0.2 ␮g/mL) in complete medium for 30 min in 37 ◦ C, protected from light. After staining, cells were washed with PBS twice and then observed with the confocal microscope (Mannheim, Germany) with a laser excitation at 543/405 nm and emission at 590/461 nm. In addition, the fluorescence intensity of Lyso-Tracker Red and Hochest 33342 was quantitatively measured using a Thermo Varioskan Flash microplate reader (Winooski, VT, USA). The abundance of lysosomes was expressed as the normalized intensity of Lyso-Tracker Red against Hochest 33342, which controlled the cell number difference among wells. FITC-dextran (20KDa, Sigma, St. Louis, USA) was employed to investigate the lysosomal membrane destabilization. Macrophages cultured on Thermo LabTek chambered borosilicate coverglass were pre-incubated with FITC-dextran (1 mg/mL) for 4 h. Cells were washed with PBS to remove residual FITC-dextran and then exposed to 10 ␮g/mL AF-SWCNTs and GOs for 24 h. After PBS wash, cells were imaged on the confocal microscope with excitation/emission at 494/518 nm. 2.9. Statistical analysis The data were expressed as the mean ± S.D., and the difference between groups was evaluated using paired Student’s t-test, with the significance level set at *p < 0.05 or **p < 0.01.

3. Results 3.1. Synthesis and characterization of acid-functionalized single-walled carbon nanotubes (AF-SWCNTs) and graphene oxides (GOs) AF-SWCNTs and GOs were synthesized following the protocol described in the methods section. The synthesized GOs were characterized with atomic force microscopy (AFM). Fig. 1A (also Fig. S3) shows the AFM image and its height profile. The measured thickness for the GO sheet was ∼1 nm, suggesting that it was a single layer sheet. The characterization of AF-SWCNTs by transmission electron microscopy (TEM) showed that AF-SWCNTs retained the structural integrity of carbon nanotubes with a typical diameter of 1–2 nm (Fig. 1B), which was consistent with the SWCNTs synthesized under the same conditions (Kam et al., 2004). Both carbon nanomaterials displayed negative surface charges as revealed by dynamic light scattering (DLS), and the zeta potentials of AF-SWCNTs and GOs in RPMI (without FBS) were −19.5 mV and −18.5 mV, and in cRPMI (with 10% FBS) were −5 mV and −7.5 mV (See Fig. S2A), respectively. The hydrodynamic diameters of AFSWCNTs and GOs were 500 nm and 355 nm (in RPMI), and 101.9 nm and 586.4 nm (in cRPMI), respectively (Fig. S2B). Surface charges of nanomaterials decreased in cRPMI compared to RPMI because of the adsorption of serum onto the surface of nanomaterials, and charges neutralization. Due to the adsorption of proteins onto the surface of nanomaterials in cRPMI, bundled nanomaterials became more dispersive and the hydrodynamic diameters were changed (Fig. S2B). Both nanomaterials were stably dispersed in culture medium. Fourier transform infrared spectroscopy (FT-IR) analyses on AF-SWCNTs and GOs showed the presence of carboxyl groups ( = 1635 cm−1 , COOH) and hydroxyl group ( = 1397 cm−1 , OH) on their surface (Fig. 1C). Raman characterization indicated a higher ratio of D-band (1347 cm−1 )/G-band (1594 cm−1 ) intensity of GOs than that of AF-SWCNTs (Fig. 1D). The ICP-MS measurement detected a negligible iron content of 0.056 wt.% for AF-SWCNTs, as reported previously (Dong et al., 2012a). The levels of metal ions in GOs were determined as 64 ppb and 61 ppb for manganese and iron, respectively. Cobalt and nickel were not detected in the samples. 3.2. Cytotoxicity of AF-SWCNTs and GOs

2.8. Evaluation of lysosomal membrane destabilization Lyso-Tracker Red (a lysosome targeting probe) and Hochest 33342 (a nucleus targeting probe) were used to label lysosome and nucleus, respectively to determine the effects of AF-SWCNTs and GOs on lysosome, and nucleus staining was

We first studied the morphological changes of nanomaterialtreated macrophages by using laser confocal microscopy and scanning electron microscopy (SEM). Confocal images of cells

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Fig. 1. Characterization of acid-functionalized single-walled carbon nanotubes (AF-SWCNTs) and graphene oxides (GOs): (A) AFM image and corresponding AFM height profile for the GO sheet. AFM measurement along the black solid line in (A) shows that the sheet is ∼1 nm thick, (B) TEM image of AF-SWCNTs, scale bar = 100 nm, and (C) FT-IR spectra of AF-SWCNTs and GOs, showing the presence of carboxyl groups (␯=1635 cm−1 , COOH) and hydroxyl group ( = 1397 cm−1 , OH), and (D) Raman spectra of AF-SWCNTs and GOs.

cultured with AF-SWCNTs and GOs showed many black nanomaterials and large vesicles inside the cells (Fig. 2A, C50 and G50), especially for cells under GO treatment. SEM images confirmed the changes by revealing multiple hollows on the cell membrane, suggesting cell membrane damage of macrophages (Fig. 2A). In parallel we performed another independent assay to measure cytotoxicity via quantifying the release of lactate dehydrogenases (LDH) into the supernatant. In our experiments, both types of nanomaterials displayed cytotoxicity in a concentration-dependent manner (Fig. 2B). GOs were more toxic when compared to AF-SWCNTs. 10 ␮g/mL GOs caused significant release of LDH from cell cytosol, which occurred only in the highest concentration (50 ␮g/mL) of AF-SWCNTs (Fig. 2B). 3.3. Autophagosome accumulation and decreased autophagic degradation in macrophages upon carbon nanomaterials exposure Recently, autophagy is considered as an important pathway of cell death (Stern et al., 2012). Although there are only a few studies on nanomaterial-induced autophagy, the autophagic process is known to be induced by metal oxides (Kenzaoui et al., 2012; Hussain and Garantziotis, 2013; Sun et al., 2012), carbon nanomaterials (Chen et al., 2012; Liu et al., 2011), gold nanomaterials (Li et al., 2010). In order to determine whether carbon nanomaterials tested

would induce autophagy or not, we first monitored the formation of autophagosomes within primary peritoneal macrophages after AF-SWCNT and GO exposure through TEM examination. Multiple characteristic double-membraned structures of autophagosomes (Mizushima et al., 2010) were revealed within cells after the exposure of AF-SWCNTs and GOs, as shown in Fig. 3A (C50 and G50). We then used the cells which transiently expressed enhanced green fluorescent protein (EGFP)-tagged LC3 (EGFP-LC3) to visualize the formation of autophagosomes, and found that both AF-SWCNTs and GOs induced the accumulation of EGFP-LC3 punctuates, showing an increased number of bright green dots within treated cells (Fig. 3B). Western-blot analysis also showed that the LC3-II/LC3-I ratio increased from 1.48 to 1.82 and 3.32 after the exposure of 50 ␮g/mL AF-SWCNTs and GOs, respectively, and in a dose-dependent manner (Fig. 3C), which further confirms autophagosome accumulation in AF-SWCNT and GO exposed macrophages. Based on these results, we conclude that both nanomaterials can induce the accumulation of autophagosomes in macrophages, but GOs are more potent than AF-SWCNTs. As LC3-II is degraded by autolysosomes, the total amount of LC3-II at any specific time point is a function of the balance between the rate of its generation and the rate of its degradation in autolysosomes (Mizushima et al., 2010). Thus autophagosome accumulation may also represent a blockage on

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Fig. 2. Cytotoxicity of AF-SWCNTs and GOs: (A) morphological change of peritoneal macrophages under exposure of 0 (VC), 50 ␮g/mL AF-SWCNTs (C50), and 50 ␮g/mL GOs (G50) for 24 h. Upper: confocal images of cells (scale bar is 25 ␮m). Lower: SEM images (scale bar is 10 ␮m). Arrows direct to vesicular, (B) cell viability was measured by LDH release. Asterisk indicates significant difference from control (0 ␮g/mL) at level *: p < 0.05, **: p< 0.01. Data are presented as the means of three experiments. Error bars represent S.D.

autophagosomal maturation and degradation. Therefore, we further analyzed the autophagic degradation of p62 (also known as SQSTM1/sequestome 1). p62 is selectively incorporated into autophagosomes through direct binding to LC3, and is preferentially degraded by autolysosomes (Bjorkoy et al., 2005; Pankiv et al., 2007). Thus total cellular expression levels of p62 inversely correlate with autophagosomal maturation and degradation (Mathew et al., 2009). We found that exposure of a well-known autophagy inducer, Rapamycin, resulted in significant degradation of p62. However, AF-SWCNTs and GOs exposure induced a dosedependent accumulation of p62 (Fig. 3C), indicating an impaired autophagic degradation. 3.4. Lysosomal accumulation of carbon nanomaterials and lysosome destabilization Given the fact that autophagosome matures through its fusion with a lysosome, forming autolysosomes and leading to the

degradation of the enclosed materials, lysosome impairment can potentially affect the autophagic degradation. Therefore, we further asked whether these two nanomaterials accumulate in lysosomes, since it is generally known lysosomal overload can lead to lysosome membrane destabilization and defects in intracellular trafficking (Futerman and van Meer, 2004), which is essential for the formation of autolysosome. We first examined the ultra-thin sections of macrophages through TEM and found the presence of a large amount of AF-SWCNTs and GOs in lysosomes (Fig. 4B and C, also Fig. S4). Thereafter, we used LysoTracker-red, a selective fluorescent acidotropic probe, to label and track acidic organelles in cells and monitor the biogenesis and pathogenesis of macrophage lysosomes under the exposure of AF-SWCNTs and GOs. As shown in Fig. 4, compared to control (Fig. 4D), cells incubated with AF-SWCNTs and GOs had decreased red fluorescence, whereas there was no change in the fluorescence intensity of Hochest 33342 (cell nucleus staining) (Fig. 4E and F). A quantitative measurement of the fluorescence intensity of LysoTracker-red showed that the normalized

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Fig. 3. Autophagosome accumulation and decreased autophagic degradation in macrophages under the exposure of carbon nanomaterials: (A) representative TEM images of macrophages under the exposure of 0 (VC), 50 ␮g/mL AF-SWCNTs (C50) and GOs (G50). Arrows directed to the double-membraned structures, a characteristic structure of autophagosomes, indicate the formation of autophagosomes, (B) representative laser confocal images of the formation of EGFP-LC3 dots (bright green) in macrophages transiently transfected with pEGFP-LC3 and then exposed to medium in the presence or the absence of carbon nanomaterials and Rapamycin. Scale bar = 10 ␮m. C. Westernblot analysis on the conversion of LC3-I to LC3-II, and the abundance of autophagic target p62 protein. Total protein was harvested and subjected to western-blot analysis with anti-LC3 or anti-p62 antibody. ␤-actin served as loading control. The level of protein expression was measured by integrated optical densitometry (IOD) using Gelpro 4.0 software. Rapamycin was used as a positive control. Error bars represent S.D. of at least three individual experiments. Asterisk indicates significant difference from control at level *p < 0.05, **p < 0.01. (For interpretation of references to color in this figure legend, the reader is referred to the web version of this article.)

fluorescence intensity decreased from 0.14 (control) to 0.09, and 0.11 for AF-SWCNT and GO treatment groups, respectively (Fig. 4J), indicating reduced lysosome abundance of the exposed cells. In addition, lysosomal membrane destabilization can be confirmed by following the diffusion into the cytosol of fluorescein isothiocyanate (FITC)-dextran preloaded in lysosomes (Thibodeau et al., 2004; Tahara et al., 2012). In our experiments, FITC-dextran was confined to the lysosomes of macrophages, as shown by the bright green spots in Fig. 4G. When the FITC-dextran preloaded macrophages were incubated with AF-SWCNTs and GOs, the number of bright spots decreased after the diffusion of the fluorescent signals throughout the cell interior (Fig. 4H and I), indicating that the lysosomal membrane was damaged, allowing the release of FITC-dextran into the cytosol. From these results, we conclude that both AF-SWCNTs and GOs accumulate in macrophage lysosomes and cause lysosome membrane destabilization. 4. Discussion In this study, primarily cultured murine peritoneal macrophages were employed as an ex vivo cellular model for toxicity testing

since they closely resemble tissue environment, and macrophages usually contribute to a first-line defence in vivo operating against foreign intruders such as nanomaterials. They are of high relevance as model systems. In addition they play important roles in the innate immune system, triggering complex immunological responses via secretion of cytokines. Previous studies of ours and others have showed that SWCNT-exposed cells might also end in an apoptosis-like death without activation of caspases, reflecting an alternative pathway (Dong et al., 2012b; Shvedova et al., 2005). Recently, a lysosome-based degradation process named cell autophagy was proposed as an important pathway of cell death (Stern et al., 2012). Various nanomaterials including metal oxides (Kenzaoui et al., 2012; Hussain and Garantziotis, 2013; Sun et al., 2012), carbon nanomaterials (Chen et al., 2012; Liu et al., 2011), gold nanomaterials (Li et al., 2010), among others, have been shown to induce cell autophagy. Autophagy is the basic catabolic mechanism that involves cell degradation of unnecessary or dysfunctional cellular components through the lysosomal machinery (Lin et al., 2013). Autophagy, if regulated, ensures the synthesis, degradation and recycling of cellular components (Lin et al., 2013). During this process, targeted cytoplasmic constituents are isolated from

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Fig. 4. Lysosomal accumulation of carbon nanomaterials and lysosome destabilization. Abundance and stability of macrophage lysosomes under 24 h exposure of 0 (A,D,G), 10 ␮g/mL of AF-SWCNTs (B,E,H) and 10 ␮g/mL of GOs (C,F,I), respectively. Upper: TEM images of macrophage lysosomes after the treatments (A,B,C). Middle: Confocal images of LysoTracker-Red (Red) and Hochest 33342 (Blue) staining overlaid with the bright field images (D,E,F). Lower: Confocal images of Dextran-FITC (20 kDa) distribution within macrophages (G,H,I). (J) Quantitative measurement of the loss of fluorescence signal from LysoTracker-Red upon carbon nanomaterial exposure. (For interpretation of references to color in this figure legend, the reader is referred to the web version of this article.)

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the rest of the cell within the autophagosomes, which are then fused with lysosomes and degraded or recycled (Patel et al., 2012). Cellular autophagic activity is usually low under basal condition, but can be markedly up-regulated by numerous stimuli such as nutrient starvation, innate immune signals, chemical reagents, and infections. Autophagy has been seen as an adaptive response to survival, whereas unstrained autophagy appears to promote cell death and morbidity (Mizushima et al., 2010; Patel et al., 2012). In practice, autophagy can be evaluated by monitoring the formation of autophagosomes qualitatively (e.g. electron microscopy, GFPLC3 cells) and quantitatively (e.g. assessing autophagy biomarker LC3-I/LC3-II transformation by western-blot) (Stern et al., 2012). Since the autophagosome is an intermediate structure in a dynamic pathway, the number of autophagosomes observed at any specific time point is a balance between the rate of their generation and the rate of their conversion into autolysosomes (Stern et al., 2012). Thus, blockade of autophagy flux and autophagy induction can both lead to autophagosome accumulation. Previous studies on nanomaterials-induced autophagy mainly focused on direct autophagy induction, while ignoring the possible role of autophagy blockade (Chen et al., 2012; Liu et al., 2011). In this study, we systematically measured the formation of autophagosomes through electron microscopy, pEGFP-LC3 transfected cells, and Westernblot. The results found that AF-SWCNTs and GOs were able to induce significant accumulation of autophagosomes (Fig. 3). However, further analysis on the autophagic degradation target p62 revealed decreased autophagic degradation, suggesting impaired autolysosomal. Additionally, our results also found the occurrence of significant lysosome impairment, manifested as overload of lysosomes by nanomaterials, decreased lysosomal stability and biogenesis. Lysosome disorders were often found to be associated with autophagy dysfunction, with blockade of autophagosome and lysosome fusion, and accumulation of autophagosomes and autophagy substrates (e.g., ubiquitinated protein aggregates) (Settembre et al., 2008). Lysosome is the main organelle to accumulate many types of nanomaterials (Dong et al., 2012a; Moore et al., 2009) and a variety of nanomaterials including carbon nanomaterials could induce lysosomal dysfunction, which has been implicated in disease pathogenesis (Stern et al., 2012). Therefore, the association of nanomaterials exposure and lysosomal dysfunction may have relevance to nanomaterial-induced toxicities (Ravikumar et al., 2010). Lysosomal overload can lead to lysosome membrane destabilization and defects in intracellular trafficking (Futerman and van Meer, 2004), which, on one hand, prevents autophagosomelysosome fusion, causing the impairment of autolysosomal degradation (Stern et al., 2012). On the other hand, carbon nanomaterial-induced lysosome membrane destabilization may occur in two forms based on the degree of membrane damage, i.e. lysosomal membrane permeabilization (LMP) and lysosomal membrane rupture (LMR). In LMP, membrane damage is limited but the release of certain molecules, including cathepsin B/D, results in apoptosis (Boya and Kroemer, 2008; Johansson et al., 2010). In deed, LMP was proposed as a potential mechanism of nanomaterialinduced toxicity in human fibroblasts and macrophages, and was found to be associated with loss of mitochondrial membrane potential and apoptosis (Sohaebuddin et al., 2010; White and DiPaola, 2009). In LMR, membrane damage is severe and various degradative enzymes in the lysosomes, such as acidic hydrolases, are released to the cytoplasm (Kagedal et al., 2001; Antunes et al., 2001) where they degrade cytoplasmic structures, leading to necrosis. Additionally, lack of functional lysosomal enzymes resulting from LMR may manifest as lysosomal storage disorders such as neurodegenerative diseases (Bellettato and Scarpa, 2010). Thus, the observations on carbon nanomaterial-induced lysosome impairment in our experiments might shed light on the mechanism of co-occurrence of apoptosis and necrosis upon the exposure of carbon nanomaterials,

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as previously reported (Dong et al., 2012a; Shvedova et al., 2005). Alternatively, as cytoskeleton plays essential roles in the membrane rearrangements and the traffic of intracellular vesicles including autophagosomes, nanomaterial-induced cytoskeleton disruption and autophagic vacuole accumulation are commonly reported in the literature. Thus the possibility that cytoskeleton disruption may also be involved in the occurrence of autophagosome accumulation cannot be ruled out (Johnson-Lyles et al., 2010; Monastyrska et al., 2009). This is in accordance with the observation that GOs and AF-SWCNTs significantly disrupted the cytoskeleton of the cells, as shown in Fig. 2A. Similar to lysosomal dysfunction, dysfunction of the autophagy pathway has also been linked to a variety of diseases including cancers, possibly by allowing the accumulation of damaged organelles, such as mitochondria, that can then induce oxidative stress, inflammation and DNA damage (White and DiPaola, 2009). In some diseases, disruption of autophagy flux may result in blockade of autophagy-mediated elimination of disease-associated proteins (e.g. amyloid beta and alpha synuclein in Parkinson’s disease) (Ravikumar et al., 2010). Thus, carbon nanomaterialinduced autophagy blockade, as revealed in this study, might suggest a potential carcinogenesis and pro-neurodegenerative activities. Simultaneously, blockade of autophagy pathway abolish its pro-survival mechanism, resulting in cytotoxicity of nanomaterials. For example, fullerenol induced cytotoxicity (mitochondrial depolarization and actin cytoskeleton disruption) was associated with increased autophagosome marker LC3-II and autophagy flux marker p62 (Johnson-Lyles et al., 2010). In our study, carbon nanomaterial-induced autophagy blockade may also be a mechanism of inflammation since autophagy negatively regulates NLRP3 inflammasome (Shi et al., 2012). It is worthy of note that, in our experiments, GOs are generally more potent than AF-SWCNTs in the induction of autophagosome accumulation and lysosomal dysfunctions, as well as cytotoxicity. Both carbon nanomaterials have similar chemical composition and profile of surface functional groups (Fig. 1C). Thus the distinct toxic potencies might be resulted from their different physical features. SWCNT consists of a graphene sheet rolled into a tube and capped by half a fullerene structure, resulting in half of the surface area inaccessible to biological molecules. Given their tubular shape, SWCNTs tend to penetrate membranes, whereas the flat shape of GOs are expected to have stronger interaction with cell membranes (Zhang et al., 2010), leading to higher effects. On the other hand, carbon-based nanomaterials tend to form microscopic bundles in environmental or biological fluids due to the high ionic strength, and the agglomeration changes the size, surface area, and sedimentation properties of the nanomaterials, which may also affect the toxicity (Zook et al., 2011). In our study, we measured the changes of hydrodynamic diameters of both carbon nanomaterials under protein-rich and protein-depleted conditions. The results found that proteins were able to assist the dispersion of nanomaterials to different degrees (Fig. S2B). In the presence of serum, GOs were larger than that of AF-SWCNTs (Fig. S2B), and it has been shown that particle size closer to the size of bacteria (micrometer range) may cause more potent response in macrophages (Doshi and Mitragotri, 2010). More work is needed to investigate in-depth the influencing factors that render these two carbon nanomaterials different toxicity profiles. The interaction of carbon nanomaterials with the autophagy and lysosomal pathways and resulting dysfunction suggest caution on their potential side effects. However, it is not necessarily always a disadvantageous scenario. The lysosomal accumulation of AF-SWCNTs and GOs can be used for medical applications. For example, lysosomal enzymes can be packed onto nanomaterials of a certain size and shape for the treatment of autophagydepleted conditions (Komatsu et al., 2006). Currently, cationic

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dendrimer-induced LMP has been used to enable lysosomal escape for cellular delivery of drugs (Nie et al., 2006). Alumina nanomaterials were used to transport bound antigen to autophagosomes of dendritic cells, while block lysosomal degradation and elicit a potent T-cell anti-tumor response (Li et al., 2011). In addition, nanomaterials with autophagy disruption activities have been proposed as new tools to monitor autophagy (Choi et al., 2011). 5. Conclusions In summary, the effects of AF-SWCNTs and GOs on cell viability, autophagy induction and lysosome destabilization were studied in primarily cultured murine peritoneal macrophages. Both carbon nanomaterials were found to induce adverse effects in cells, and GOs were more potent than AF-SWCNTs. Analyses on autophagosome, lysosome, as well as p62 protein degradation in carbon nanomaterial-exposed cells suggested that both carbon nanomaterials were able to induce autophagosome accumulation, decreased autophagic degradation and lysosomal impairment. The results may shed light on the potential toxic mechanism of carbon nanomaterials and suggest caution on their utilization, while also provide new insights to their potential medical applications. Conflict of interest statement The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. Acknowledgements This work was supported by the National Basic Research Program of China (Nos. 2009CB421605, 2011CB936001), the National Natural Science Foundation of China (21277158, 21177138, 21077124). The authors thank Dr. Wei Zhang of the College of Life Sciences, Beijing Normal University for providing pEGFP-LC3 plasmid. The authors also thank the lab of Bio-imaging, the Institute of Biophysics for the TEM work and specifically Sun Lei and Liu Yanrong for their assistance in TEM sample preparation and photographing. Briefing: AF-SWCNT and GO induce autophagosome accumulation and lysosome damage Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.toxlet. 2013.06.208. References Agarwal, S., Zhou, X.Z., Ye, F., He, Q.Y., Chen, G.C.K., Soo, J., Boey, F., Zhang, H., Chen, P., 2010. Interfacing live cells with nanocarbon substrates. Langmuir 26, 2244–2247. Antunes, F., Cadenas, E., Brunk, U.T., 2001. Apoptosis induced by exposure to a low steady-state concentration of H2 O2 is a consequence of lysosomal rupture. Biochemical Journal 356, 549–555. Ball, P., 2001. Roll up for the revolution. Nature 414, 142–144. Bellettato, C.M., Scarpa, M., 2010. Pathophysiology of neuropathic lysosomal storage disorders. Journal of Inherited Metabolic Disease 33, 347–362. Bianco, A., Kostarelos, K., Prato, M., 2005. Applications of carbon nanotubes in drug delivery. Current Opinion in Chemical Biology 9, 674–679. Bjorkoy, G., Lamark, T., Brech, A., Outzen, H., Perander, M., Overvatn, A., Stenmark, H., Johansen, T., 2005. p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. Journal of Cell Biology 171, 603–614. Boya, P., Kroemer, G., 2008. Lysosomal membrane permeabilization in cell death. Oncogene 27, 6434–6451. Caravan, P., Ellison, J.J., McMurry, T.J., Lauffer, R.B., 1999. Gadolinium(III) chelates as MRI contrast agents: structure, dynamics, and applications. Chemical Reviews 99, 2293–2352.

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