Arch Toxicol DOI 10.1007/s00204-014-1303-x
Molecular Toxicology
Toxic response of graphene nanoplatelets in vivo and in vitro Eun‑Jung Park · Gwang‑Hee Lee · Beom Seok Han · Byoung‑Seok Lee · Somin Lee · Myung‑Haing Cho · Jae‑Ho Kim · Dong‑Wan Kim
Received: 3 April 2014 / Accepted: 17 June 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract With the development of nanotechnology, myriad types of novel materials have been discovered at the nanoscale, among which the most interesting material is graphene. However, the toxicity data available on graphene are extremely limited. In this study, we explored toxic response of commercially available graphene nanoplatelets (GNPs) in vivo and in vitro. The GNPs used in this study had a high surface area and feature considerably few defects. In mice, GNPs (2.5 and 5 mg/kg) remained in the lung until 28 days after a single instillation, and the secretion of inflammatory cytokines reached the maximal Eun-Jung Park and Gwang-Hee Lee have contributed equally to this work.
level at Day 14 and then decreased over time. In vitro study using BEAS-2B cells, a human bronchial epithelial cell line, GNPs located within autophagosome-like vacuoles 24 h after exposure. The GNPs (2.5, 5, 10, and 20 μg/mL) also dose-dependently reduced cell viability, which was accompanied by an increase in the portion of cells in the subG1 and S phases. Moreover, the GNPs down-regulated the generation of reactive oxygen species, suppressed ATP production, caused mitochondria damage, and elevated the levels of autophagy-related proteins. Based on these results, we suggest that GNPs provoked a subchronic inflammatory response in mice and that GNPs induced autophagy accompanying apoptosis via mitochondria damage in vitro.
Electronic supplementary material The online version of this article (doi:10.1007/s00204-014-1303-x) contains supplementary material, which is available to authorized users.
Keywords Graphene · Nanoplatelets · Toxicity · Autophagy · Mitochondria
E.-J. Park (*) · J.-H. Kim Department of Molecular Science and Technology, Ajou University, Suwon 443‑749, Korea e-mail: [email protected]
Introduction
G.-H. Lee · D.-W. Kim (*) School of Civil, Environmental, and Architectural Engineering, Korea University, Seoul 136‑713, Korea e-mail: [email protected] B. S. Han Hoseo Toxicological Research Center, Hoseo University, Asan 336‑795, Korea B.-S. Lee Toxicologic Pathology Center, Korea Institute of Toxicology, Jeongeup 580‑185, Korea S. Lee · M.-H. Cho College of Veterinary Medicine, Seoul National University, Seoul 151‑742, Korea
With the development of nanotechnology, diverse types of novel materials at the nanoscale have been discovered, and one of these materials is graphene. Geim and Novoselov won the 2010 Nobel Prize in Physics for their work on graphene, and the discovery of graphene became a new driving force in the growth of nanoindustry. Graphene is a crystalline form of carbon in which the atoms are arranged in a regular hexagonal pattern. Graphene is a strong and light material that conducts heat and electricity extremely well, as carbon nanotubes do (Bussy et al. 2013). Since graphene was first isolated in 2004, its properties have been studied extensively, and graphene and graphene-based nanomaterials are today applied in numerous fields for purposes including the development of electronics, energy storage,
13
biosensors, and biomedicine (Zhang et al. 2012; Brownson and Banks 2010). Concurrently, the potential toxic effects of graphene on the environment and on human health have attracted considerable attention among both toxicologists and engineers. Previous reports have suggested that pristine graphene exhibits severe toxicity in various biological system, but that surface functionalized-graphene is minimally toxic (Sasidharan et al. 2011, 2012; Yang et al. 2011, 2013; Lammel et al. 2013). Moreover, graphene induced apoptosis by lowering the mitochondrial membrane potential and increasing the levels of intracellular reactive oxygen species (ROS) in macrophages (Li et al. 2012; Sasidharan et al. 2011, 2012). Sheet-type graphene induced Th2-type inflammatory responses (Wang et al. 2013), whereas platelet-type graphene drove inflammogenicity in vivo and in vitro (Schinwald et al. 2012). Furthermore, graphene oxide, one of the most important graphene derivatives, also decreased cell viability, damaged the cell membrane and concomitantly increased ROS levels (Chang et al. 2011; Gurunathan et al. 2013), and led to necroptosis by activating Toll-like receptor 4 signaling in macrophages (Qu et al. 2013; Linkermann et al. 2012; Wu et al. 2012). Toxic responses elicited by nanomaterials differ according to the unique properties of the nanomaterials, including their size, shape, structure, surface functional groups, coatings, and dispersion state, as well as the properties of the biological system used in the experiment. In this study, we characterized commercially available graphene nanoplatelets (GNPs) and then explored the toxic response of these GNPs in vivo and in vitro.
Arch Toxicol
Potential and Particle Size Analyzer (ELSZ-2, Otsuka Electronics Korea Co. Ltd). In vivo sample preparation Six-week-old male ICR mice (27–28 g, OrientBio, Seongnam, Korea) were acclimatized for 1 week before the start of the study at constant temperature of 23 ± 3 °C, relative humidity of 50 ± 10 %, a 12-h light/dark cycle with a light of intensity 150–300 lx, and ventilation of 10–20 times/h. Temperature and relative humidity were monitored and recorded daily. After acclimation, GNPs (900 μg/mL in PBS) were delivered using a 24-gauge catheter at a dose of 2.5 and 5 mg/kg by intratracheal instillation under light tiletamine anesthesia. The control group was treated with autoclaved PBS, and the animals (10 mice/group) were euthanized at 1, 7, 14, and 28 days after treatment. Bronchoalveolar lavage (BAL) fluid was obtained by cannulating the trachea and lavaging the lungs with 1 mL of cold sterile (Ca2+ plus Mg2+)-free PBS (0.15 M, pH 7.2) (Park et al. 2009). The BAL fluid (500–600 μL) was centrifuged at 3,000 rpm for 10 min, and the supernatant was used for cytokine analysis (n = 3, 2 mice/test sample). In addition, approximately 1.2 mL of blood per mouse was collected from the saphenous vein. The whole blood was centrifuged at 3,000 rpm for 10 min to obtain serum for cytokine analysis. This experiment (IACUC No. 2012-0007) was assessed by the Institutional Animal Care and Committee (IACUC) of Ajou University (Suwon, Korea) and performed in accordance with the “Guide for the Care and Use of Laboratory Animals”, an ILAR publication. Histopathological analysis
Materials and methods Suspension and characterization of GNPs Pristine GNPs (Hanwha Nanotech, Korea) were loaded in deionized water (DW) at a concentration of 1 mg/mL and sonicated for 1 h to stably disperse the GNPs. The morphology and surface properties of GNPs were investigated using transmission electron microscopy (TEM, Tecnai G2 F30 S-Twin, FEI, Hilsboro, OR, USA), and using a Raman spectrometer (NRS-3100, Jasco, Tokyo, Japan) and a Fourier-transform infrared spectrometer (FT-IR, Bruker IFS-66/S, Bruker, Karlsruhe, Germany). The specific surface areas of the GNPs were also estimated using a Brunauer–Emmett–Teller surface area analyzer (BET, Belsorp mini II, BEL Japan Inc., Osaka, Japan). In addition, the surface charge and hydrodynamic length in vehicles were characterized by using the Zeta
13
Lung tissue was harvested from four mice per group. The lung was fixed with 10 % neutral buffered formalin and processed using routine histological techniques. After paraffin embedding, 3-μm sections were cut and stained with hematoxylin and eosin for histopathological evaluation. Cytokine analysis The concentration of each cytokine (IL-1β, TNF alpha, IL-6, GM-CSF, TGFβ, and MCP-1) was determined using commercially available enzyme-linked immunosorbent assay (ELISA) kits as described previously (eBioscience, Park et al. 2009). Briefly, each well in the 96-well immunoplate was coated with capture antibody and incubated overnight at 4 °C. After blocking with assay diluents, the BAL fluid, serum, and standard antibody was added to each well. Following, the assay was performed according to manufacturer’s manual and finally the absorbance was measured at
Arch Toxicol
450 nm using an ELISA plate reader (Molecular Devices). The amount of each cytokine was calculated from the linear portion of the standard curve which was generated in the same condition. Cell culture BEAS-2B cells, a human bronchial epithelial cell line, were purchased from American Type Culture Collection (ATCC, VA, USA). BEAS-2B cells were maintained in DMEM/F12 (Life Technologies, Seoul, Korea) containing 10 % fetal bovine serum (FBS), penicillin 100 IU/ml, and streptomycin 100 µg/ml at 37 °C in a 5 % CO2 humidified incubator. Cell viability Cell viability was measured by MTT (3-(4-5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma-Aldrich, MO, USA) assay as described previously (Park et al. 2014). Briefly, cells (1 × 104 or 5 × 103 cells/100 μL/well for 24 h or 72 h, respectively) were seeded on 96-well plates. After stabilization overnight, cells were exposed to 2.5, 5, 10, and 20 μg/mL of GNPs for the designated times, and then MTT solution (2 mg/mL, 40 μL/well) was added to each well. After a further incubation for 3 h at 37 °C, the absorbance was quantified at 540 nm using the microplate spectrophotometer system (Molecular Devices, Sunnyvale, CA, USA). The viability of the treatment group was expressed as a percentage of the control group, which was considered as 100 %. LDH assay The LDH levels were determined using commercially available LDH assay kits (Biovision, CA, USA) according to the manufacturer’s instructions. Briefly, cells (2 × 104 or 1 × 104 cells/100 μL/well for 24 or 72 h, respectively) were seeded in a 96-well plate. After overnight, GNPs were added at the designated concentration and the cells were incubated for 24 or 72 h. Then, the supernatants (10 μL/ well) were transferred into a new 96-well plate, and LDH reaction solution (100 μL) was added to each well. After further incubation for 30 min at room temperature (RT), the absorbance was quantified at 450 nm using the microplate spectrophotometer system (MolecularDevices). Cell cycle analysis At the end of exposure, total cells were fixed with 70 % ethanol and stained with propidium iodide and digested with RNAse (Sigma-Aldrich). Then, the cell cycle was analyzed by measuring the DNA content using the FACSCalibur system and CellQuest software (BD Biosciences, Franklin Lakes, NJ, USA) (Park et al. 2013).
ROS and NO generation Generation of ROS and NO were performed as reported previously (Park et al. 2014). Briefly, cells were treated with different concentrations of GNPs for 24 or 72 h, and then further incubated with 5 μM carboxy-2′7′dichlorodihydrofluorescein diacetate (H2DCFDA, Invitrogen, Carlsbad, CA, USA) for 30 min. The number of cells generating ROS was counted using the FACSCalibur system and analyzed with CellQuest software (BD Biosciences). For NO assay, cells were incubated in the absence or the presence of GNPs for 24 h (1 × 106 cells/ mL) or 72 h (5 × 105 cells/mL). The supernatants (100 µL/ well) were transferred to new 96-well plates and were tested using a NO detection kit (iNtRON Biotech, Gyeonggi-do, Korea). The values were calculated by measuring the absorbance at 540 nm using the microplate spectrophotometer system (Molecular Devices, Sunnyvale, CA, USA). ATP measurement Cells were seeded at a density of 1 × 104 cells/well (24 h) or 5 × 103 cells/well (72 h) into white 96-well plates and stabilized overnight (Park et al. 2014). Cells were exposed to 2.5, 5, 10, and 20 μg/mL of GNPs, and then plate was incubated for 24 or 72 h. At the end of exposure, CellTiter-Glo® Reagent (Promega, Fitchburg, WI, USA) was added as a 1:1 volume ratio, and the plate was incubated at RT for 10 min. The luminescence value was measured using a microplate luminometer (Berthold Detection Systems, Berthold Technologies GmbH & Co. KG, Bad Wilbad, Germany). Protein expression Cell pellet was homogenized with a protein extraction solution (iNtRON Biotech) and the lysates were acquired from centrifugation at 13,000 rpm for 30 min. The protein concentration was measured by the bicinchonic acid method (Sigma-Aldrich), and equal amounts of protein were separated on a 12–15 % sodium dodecyl sulfate– polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane (Hybond ECL, Amersham Pharmacia Biotech, NJ, USA). Blots were blocked for 1 h at RT with 5 % nonfat dried milk in tris-buffered saline solution containing 0.05 % Tween-20 (TBST). The membranes were immunoblotted with primary specific antibodies (1:1,000 dilution): rabbit polyclonal antibody for microtubule-associated protein 1 light chain 3 (LC3B) and cytochrome C (Cell Signaling Technology, MA, USA), beclin 1 (Santa Cruz Biotech, CA, USA), and autophagy protein 5 (ATG5, abcam, MA, USA), mouse monoclonal antibody for p62 (abcam) and Bcl-2 (Santa Cruz Biotech), rabbit monoclonal antibody for Bcl-2-associated X protein
13
(BAX, Santa Cruz Biotech), and goat polyclonal antibody for β-actin (Santa Cruz Biotech). The blots were then incubated with the corresponding conjugated anti-rabbit, anti-mouse or anti-goat immunoglobulin G-horseradish peroxidase (1:1,000 dilution, Santa Cruz Biotech). Immunoreactive proteins were detected with the ECL Western blotting detection system. Immunohistochemistry Cells (1 × 104) were seeded in two-well chamber slides and incubated for 24 h with or without GNPs (20 μg/mL). Cells were fixed in 4 % paraformaldehyde for 15 min at RT and then fixed again with ice-cold methanol at −20 °C. After blocking with 3 % BSA in TBST for 1 h, the cells were incubated in a 1:100 dilution of primary antibody (lysosomal-associated membrane protein (LAMP)-2, Hsp27, Hsp70, and GM130 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and LC3B and calnexin (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. After washing, the cells were incubated in a 1:100 dilution of Alexa Fluor 555- or 488-conjugated secondary antibody for 2 h at RT. Mitochondria were labeled with MitoTracker® Deep Red FM (Invitrogen) for 30 min, respectively, and then the cells were fixed in 4 % paraformaldehyde for 15 min at 37 °C. After washing, coverslips were mounted using FLUOROSHIELDT mounting medium and DAPI (ImmunoBioScience, Mukilteo, WA, USA). The slides were visualized using a fluorescent microscope (Carl Zeiss). Morphological changes For analysis of morphological changes, cells were exposed for 24 h with GNPs (20 µg/ml). After washing with PBS, cells were immediately fixed in 2 % glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 2 h (Park et al. 2014). Then, the cells were stained for 30 min in 0.5 % aqueous uranyl acetate, dehydrated in graded ethanol solutions, and embedded in Spurr’s resin. Thin sections were cut using an ultramicrotome (MT-X, RMC, Tucson, AZ, USA), stained with 2 % uranyl acetate and Reynolds’s lead citrate, and examined with a LIBRA 120 transmission electron microscope (Zeiss, Oberkochen, Germany) at an accelerating voltage of 80 kV. Statistical analysis Statistical analyses were performed using Student’s t test (Graphpad Software, San Diego, CA, USA) and one-way ANOVA test followed by Tukey’s post hoc pairwise comparison. Asterisks (*) indicated statistically significant differences to the control group, *p
Molecular Toxicology
Toxic response of graphene nanoplatelets in vivo and in vitro Eun‑Jung Park · Gwang‑Hee Lee · Beom Seok Han · Byoung‑Seok Lee · Somin Lee · Myung‑Haing Cho · Jae‑Ho Kim · Dong‑Wan Kim
Received: 3 April 2014 / Accepted: 17 June 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract With the development of nanotechnology, myriad types of novel materials have been discovered at the nanoscale, among which the most interesting material is graphene. However, the toxicity data available on graphene are extremely limited. In this study, we explored toxic response of commercially available graphene nanoplatelets (GNPs) in vivo and in vitro. The GNPs used in this study had a high surface area and feature considerably few defects. In mice, GNPs (2.5 and 5 mg/kg) remained in the lung until 28 days after a single instillation, and the secretion of inflammatory cytokines reached the maximal Eun-Jung Park and Gwang-Hee Lee have contributed equally to this work.
level at Day 14 and then decreased over time. In vitro study using BEAS-2B cells, a human bronchial epithelial cell line, GNPs located within autophagosome-like vacuoles 24 h after exposure. The GNPs (2.5, 5, 10, and 20 μg/mL) also dose-dependently reduced cell viability, which was accompanied by an increase in the portion of cells in the subG1 and S phases. Moreover, the GNPs down-regulated the generation of reactive oxygen species, suppressed ATP production, caused mitochondria damage, and elevated the levels of autophagy-related proteins. Based on these results, we suggest that GNPs provoked a subchronic inflammatory response in mice and that GNPs induced autophagy accompanying apoptosis via mitochondria damage in vitro.
Electronic supplementary material The online version of this article (doi:10.1007/s00204-014-1303-x) contains supplementary material, which is available to authorized users.
Keywords Graphene · Nanoplatelets · Toxicity · Autophagy · Mitochondria
E.-J. Park (*) · J.-H. Kim Department of Molecular Science and Technology, Ajou University, Suwon 443‑749, Korea e-mail: [email protected]
Introduction
G.-H. Lee · D.-W. Kim (*) School of Civil, Environmental, and Architectural Engineering, Korea University, Seoul 136‑713, Korea e-mail: [email protected] B. S. Han Hoseo Toxicological Research Center, Hoseo University, Asan 336‑795, Korea B.-S. Lee Toxicologic Pathology Center, Korea Institute of Toxicology, Jeongeup 580‑185, Korea S. Lee · M.-H. Cho College of Veterinary Medicine, Seoul National University, Seoul 151‑742, Korea
With the development of nanotechnology, diverse types of novel materials at the nanoscale have been discovered, and one of these materials is graphene. Geim and Novoselov won the 2010 Nobel Prize in Physics for their work on graphene, and the discovery of graphene became a new driving force in the growth of nanoindustry. Graphene is a crystalline form of carbon in which the atoms are arranged in a regular hexagonal pattern. Graphene is a strong and light material that conducts heat and electricity extremely well, as carbon nanotubes do (Bussy et al. 2013). Since graphene was first isolated in 2004, its properties have been studied extensively, and graphene and graphene-based nanomaterials are today applied in numerous fields for purposes including the development of electronics, energy storage,
13
biosensors, and biomedicine (Zhang et al. 2012; Brownson and Banks 2010). Concurrently, the potential toxic effects of graphene on the environment and on human health have attracted considerable attention among both toxicologists and engineers. Previous reports have suggested that pristine graphene exhibits severe toxicity in various biological system, but that surface functionalized-graphene is minimally toxic (Sasidharan et al. 2011, 2012; Yang et al. 2011, 2013; Lammel et al. 2013). Moreover, graphene induced apoptosis by lowering the mitochondrial membrane potential and increasing the levels of intracellular reactive oxygen species (ROS) in macrophages (Li et al. 2012; Sasidharan et al. 2011, 2012). Sheet-type graphene induced Th2-type inflammatory responses (Wang et al. 2013), whereas platelet-type graphene drove inflammogenicity in vivo and in vitro (Schinwald et al. 2012). Furthermore, graphene oxide, one of the most important graphene derivatives, also decreased cell viability, damaged the cell membrane and concomitantly increased ROS levels (Chang et al. 2011; Gurunathan et al. 2013), and led to necroptosis by activating Toll-like receptor 4 signaling in macrophages (Qu et al. 2013; Linkermann et al. 2012; Wu et al. 2012). Toxic responses elicited by nanomaterials differ according to the unique properties of the nanomaterials, including their size, shape, structure, surface functional groups, coatings, and dispersion state, as well as the properties of the biological system used in the experiment. In this study, we characterized commercially available graphene nanoplatelets (GNPs) and then explored the toxic response of these GNPs in vivo and in vitro.
Arch Toxicol
Potential and Particle Size Analyzer (ELSZ-2, Otsuka Electronics Korea Co. Ltd). In vivo sample preparation Six-week-old male ICR mice (27–28 g, OrientBio, Seongnam, Korea) were acclimatized for 1 week before the start of the study at constant temperature of 23 ± 3 °C, relative humidity of 50 ± 10 %, a 12-h light/dark cycle with a light of intensity 150–300 lx, and ventilation of 10–20 times/h. Temperature and relative humidity were monitored and recorded daily. After acclimation, GNPs (900 μg/mL in PBS) were delivered using a 24-gauge catheter at a dose of 2.5 and 5 mg/kg by intratracheal instillation under light tiletamine anesthesia. The control group was treated with autoclaved PBS, and the animals (10 mice/group) were euthanized at 1, 7, 14, and 28 days after treatment. Bronchoalveolar lavage (BAL) fluid was obtained by cannulating the trachea and lavaging the lungs with 1 mL of cold sterile (Ca2+ plus Mg2+)-free PBS (0.15 M, pH 7.2) (Park et al. 2009). The BAL fluid (500–600 μL) was centrifuged at 3,000 rpm for 10 min, and the supernatant was used for cytokine analysis (n = 3, 2 mice/test sample). In addition, approximately 1.2 mL of blood per mouse was collected from the saphenous vein. The whole blood was centrifuged at 3,000 rpm for 10 min to obtain serum for cytokine analysis. This experiment (IACUC No. 2012-0007) was assessed by the Institutional Animal Care and Committee (IACUC) of Ajou University (Suwon, Korea) and performed in accordance with the “Guide for the Care and Use of Laboratory Animals”, an ILAR publication. Histopathological analysis
Materials and methods Suspension and characterization of GNPs Pristine GNPs (Hanwha Nanotech, Korea) were loaded in deionized water (DW) at a concentration of 1 mg/mL and sonicated for 1 h to stably disperse the GNPs. The morphology and surface properties of GNPs were investigated using transmission electron microscopy (TEM, Tecnai G2 F30 S-Twin, FEI, Hilsboro, OR, USA), and using a Raman spectrometer (NRS-3100, Jasco, Tokyo, Japan) and a Fourier-transform infrared spectrometer (FT-IR, Bruker IFS-66/S, Bruker, Karlsruhe, Germany). The specific surface areas of the GNPs were also estimated using a Brunauer–Emmett–Teller surface area analyzer (BET, Belsorp mini II, BEL Japan Inc., Osaka, Japan). In addition, the surface charge and hydrodynamic length in vehicles were characterized by using the Zeta
13
Lung tissue was harvested from four mice per group. The lung was fixed with 10 % neutral buffered formalin and processed using routine histological techniques. After paraffin embedding, 3-μm sections were cut and stained with hematoxylin and eosin for histopathological evaluation. Cytokine analysis The concentration of each cytokine (IL-1β, TNF alpha, IL-6, GM-CSF, TGFβ, and MCP-1) was determined using commercially available enzyme-linked immunosorbent assay (ELISA) kits as described previously (eBioscience, Park et al. 2009). Briefly, each well in the 96-well immunoplate was coated with capture antibody and incubated overnight at 4 °C. After blocking with assay diluents, the BAL fluid, serum, and standard antibody was added to each well. Following, the assay was performed according to manufacturer’s manual and finally the absorbance was measured at
Arch Toxicol
450 nm using an ELISA plate reader (Molecular Devices). The amount of each cytokine was calculated from the linear portion of the standard curve which was generated in the same condition. Cell culture BEAS-2B cells, a human bronchial epithelial cell line, were purchased from American Type Culture Collection (ATCC, VA, USA). BEAS-2B cells were maintained in DMEM/F12 (Life Technologies, Seoul, Korea) containing 10 % fetal bovine serum (FBS), penicillin 100 IU/ml, and streptomycin 100 µg/ml at 37 °C in a 5 % CO2 humidified incubator. Cell viability Cell viability was measured by MTT (3-(4-5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma-Aldrich, MO, USA) assay as described previously (Park et al. 2014). Briefly, cells (1 × 104 or 5 × 103 cells/100 μL/well for 24 h or 72 h, respectively) were seeded on 96-well plates. After stabilization overnight, cells were exposed to 2.5, 5, 10, and 20 μg/mL of GNPs for the designated times, and then MTT solution (2 mg/mL, 40 μL/well) was added to each well. After a further incubation for 3 h at 37 °C, the absorbance was quantified at 540 nm using the microplate spectrophotometer system (Molecular Devices, Sunnyvale, CA, USA). The viability of the treatment group was expressed as a percentage of the control group, which was considered as 100 %. LDH assay The LDH levels were determined using commercially available LDH assay kits (Biovision, CA, USA) according to the manufacturer’s instructions. Briefly, cells (2 × 104 or 1 × 104 cells/100 μL/well for 24 or 72 h, respectively) were seeded in a 96-well plate. After overnight, GNPs were added at the designated concentration and the cells were incubated for 24 or 72 h. Then, the supernatants (10 μL/ well) were transferred into a new 96-well plate, and LDH reaction solution (100 μL) was added to each well. After further incubation for 30 min at room temperature (RT), the absorbance was quantified at 450 nm using the microplate spectrophotometer system (MolecularDevices). Cell cycle analysis At the end of exposure, total cells were fixed with 70 % ethanol and stained with propidium iodide and digested with RNAse (Sigma-Aldrich). Then, the cell cycle was analyzed by measuring the DNA content using the FACSCalibur system and CellQuest software (BD Biosciences, Franklin Lakes, NJ, USA) (Park et al. 2013).
ROS and NO generation Generation of ROS and NO were performed as reported previously (Park et al. 2014). Briefly, cells were treated with different concentrations of GNPs for 24 or 72 h, and then further incubated with 5 μM carboxy-2′7′dichlorodihydrofluorescein diacetate (H2DCFDA, Invitrogen, Carlsbad, CA, USA) for 30 min. The number of cells generating ROS was counted using the FACSCalibur system and analyzed with CellQuest software (BD Biosciences). For NO assay, cells were incubated in the absence or the presence of GNPs for 24 h (1 × 106 cells/ mL) or 72 h (5 × 105 cells/mL). The supernatants (100 µL/ well) were transferred to new 96-well plates and were tested using a NO detection kit (iNtRON Biotech, Gyeonggi-do, Korea). The values were calculated by measuring the absorbance at 540 nm using the microplate spectrophotometer system (Molecular Devices, Sunnyvale, CA, USA). ATP measurement Cells were seeded at a density of 1 × 104 cells/well (24 h) or 5 × 103 cells/well (72 h) into white 96-well plates and stabilized overnight (Park et al. 2014). Cells were exposed to 2.5, 5, 10, and 20 μg/mL of GNPs, and then plate was incubated for 24 or 72 h. At the end of exposure, CellTiter-Glo® Reagent (Promega, Fitchburg, WI, USA) was added as a 1:1 volume ratio, and the plate was incubated at RT for 10 min. The luminescence value was measured using a microplate luminometer (Berthold Detection Systems, Berthold Technologies GmbH & Co. KG, Bad Wilbad, Germany). Protein expression Cell pellet was homogenized with a protein extraction solution (iNtRON Biotech) and the lysates were acquired from centrifugation at 13,000 rpm for 30 min. The protein concentration was measured by the bicinchonic acid method (Sigma-Aldrich), and equal amounts of protein were separated on a 12–15 % sodium dodecyl sulfate– polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane (Hybond ECL, Amersham Pharmacia Biotech, NJ, USA). Blots were blocked for 1 h at RT with 5 % nonfat dried milk in tris-buffered saline solution containing 0.05 % Tween-20 (TBST). The membranes were immunoblotted with primary specific antibodies (1:1,000 dilution): rabbit polyclonal antibody for microtubule-associated protein 1 light chain 3 (LC3B) and cytochrome C (Cell Signaling Technology, MA, USA), beclin 1 (Santa Cruz Biotech, CA, USA), and autophagy protein 5 (ATG5, abcam, MA, USA), mouse monoclonal antibody for p62 (abcam) and Bcl-2 (Santa Cruz Biotech), rabbit monoclonal antibody for Bcl-2-associated X protein
13
(BAX, Santa Cruz Biotech), and goat polyclonal antibody for β-actin (Santa Cruz Biotech). The blots were then incubated with the corresponding conjugated anti-rabbit, anti-mouse or anti-goat immunoglobulin G-horseradish peroxidase (1:1,000 dilution, Santa Cruz Biotech). Immunoreactive proteins were detected with the ECL Western blotting detection system. Immunohistochemistry Cells (1 × 104) were seeded in two-well chamber slides and incubated for 24 h with or without GNPs (20 μg/mL). Cells were fixed in 4 % paraformaldehyde for 15 min at RT and then fixed again with ice-cold methanol at −20 °C. After blocking with 3 % BSA in TBST for 1 h, the cells were incubated in a 1:100 dilution of primary antibody (lysosomal-associated membrane protein (LAMP)-2, Hsp27, Hsp70, and GM130 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and LC3B and calnexin (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. After washing, the cells were incubated in a 1:100 dilution of Alexa Fluor 555- or 488-conjugated secondary antibody for 2 h at RT. Mitochondria were labeled with MitoTracker® Deep Red FM (Invitrogen) for 30 min, respectively, and then the cells were fixed in 4 % paraformaldehyde for 15 min at 37 °C. After washing, coverslips were mounted using FLUOROSHIELDT mounting medium and DAPI (ImmunoBioScience, Mukilteo, WA, USA). The slides were visualized using a fluorescent microscope (Carl Zeiss). Morphological changes For analysis of morphological changes, cells were exposed for 24 h with GNPs (20 µg/ml). After washing with PBS, cells were immediately fixed in 2 % glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 2 h (Park et al. 2014). Then, the cells were stained for 30 min in 0.5 % aqueous uranyl acetate, dehydrated in graded ethanol solutions, and embedded in Spurr’s resin. Thin sections were cut using an ultramicrotome (MT-X, RMC, Tucson, AZ, USA), stained with 2 % uranyl acetate and Reynolds’s lead citrate, and examined with a LIBRA 120 transmission electron microscope (Zeiss, Oberkochen, Germany) at an accelerating voltage of 80 kV. Statistical analysis Statistical analyses were performed using Student’s t test (Graphpad Software, San Diego, CA, USA) and one-way ANOVA test followed by Tukey’s post hoc pairwise comparison. Asterisks (*) indicated statistically significant differences to the control group, *p
