Drp1-Dependent Mitochondrial Fission Mediates Toxicity of Positively

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Drp1-dependent mitochondrial fission mediates toxicity of positively charged graphene in microglia Shefang Ye, Peiyan Yang, Keman Cheng, Tong Zhou, Yange Wang, Zhenqing Hou, Yuanqing Jiang, and Lei Ren ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.5b00465 • Publication Date (Web): 22 Mar 2016 Downloaded from http://pubs.acs.org on March 28, 2016

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ACS Biomaterials Science & Engineering

Drp1-dependent mitochondrial fission mediates toxicity of positively charged graphene in microglia

Shefang Ye

†*

, Peiyan Yang §, Keman Cheng †, Tong Zhou †, Yange Wang †,

Zhenqing Hou †, Yuanqin Jiang §, Lei Ren † ‡ †

Research Center of Biomedical Engineering, Department of Biomaterials, College of

Materials, Xiamen University, Xiamen 361005, PR China §

Department of Surgery, First Affiliated Hospital of Xiamen University, Xiamen,

University, 361003, PR China ‡

Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine

Biological Resources, Xiamen 361005, PR China

Corresponding Author: [email protected]

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ABSTRACT The unique physicochemical properties of graphene and its derivatives enable their application in the diagnostics and therapy of central nervous system (CNS) diseases. However, the potential impacts of surface properties of functionalized graphene on microglia remain poorly understood. Herein, we used graphene oxides (GO), polyethylene glycol (PEG)- and polyethylenimine (PEI)-functionalized GO, which possess different surface charges, to investigate their effects on microglia by focusing on mitochondrial dynamics. The positively charged GO-PEI was found to promote mitochondrial fission as observed in BV-2 cells with mitochondria labeled by DsRed2-mito, indicating that alterations in mitochondrial dynamics depend on the surface properties of graphene. Concurrent to mitochondrial fragmentation, treatment with positively charged GO-PEI induced an increase in mitochondrial recruitment of dynamin-related protein (Drp1). Additionally, GO-PEI treatment also led to apoptotic and autophagic cell death. However, Drp1 silencing by small interfering RNA (siRNA) could effectively attenuate GO-PEI-induced apoptotic and autophagic cell death,

indicating

that

mitochondrial

fragmentation

occurs

upstream

of

GO-PEI-mediated toxicity in microglia. Overall, our study indicated that positively charged GO-PEI might cause deleterious influence on the central immune homeostasis by Drp1-dependent mitochondrial fragmentation, and provide the strategies for the rational design of graphene-based materials in neuroscience.


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Keywords: Functionalized graphene, Drp1, mitochondrial dynamics, microglia, neurotoxicity

1. INTRODUCTION As a two-dimensional (2D) monolayer sheet of sp2-bonded carbon atoms, graphene is emerging as one of the most fascinating materials in nanoscience and nanotechnology.1 The distinct physicochemical properties of graphene, such as the large surface area, versatility in surface modification, unique photophysical features, and potential biocompatibility have brought it under the spotlight, 2 and made it a promising candidate for various biomedical applications, such as biosensors, 2,3 drug and gene delivery,4 photothermal therapy (PTT),5 electrical stimulation of cells,6 and cell imaging and in vivo tracing.7 In addition, great efforts has also been devoted to exploring graphene-based materials as interesting substrates or scaffolds to direct neural stem cell differentiation,8 promote neuron adhesion and neurite outgrowth.9 Recent reports have indicated that graphene-based materials might serve as ideal tools for neurostimulation and neuromonitoring, which are necessary for diagnostics and treatments in clinical practice.10 In light of extensive biomedical applications of graphene-based materials in neuroscience, a mechanistic understanding of interactions between graphene and immune cells in the brain has become imperative. Microglia, the resident immune cells in the central nervous system (CNS), represent the frontline of defense against invading foreign objects when exposed 3 ACS Paragon Plus Environment


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through either biomedical applications or environmental absorption.11,12 In this case, microglia act as primary responders that initiate subsequent in vivo immune responses, which pose profound impact on the performance of graphene-based materials. Although graphene and its derivatives look attractive, they are also plagued with

their potentially harmful health impacts.13,14 For example, in vivo studies

showed that functionalized graphene can induce pulmonary inflammation and granulomas in the lungs.15 In vitro studies showed that functionalized graphene might be cytotoxic and inhibit cell proliferation,16 induce oxidative stress and cytokine production,17,18 trigger apoptotic and autophagic cell death,19,20 and perturb cellular pathways.20 Although the molecular events leading to the graphene-mediated toxicity remain obscure, growing evidence indicates that mitochondrial dysfunctions involving depolarized mitochondrial membrane potential (∆Ψm) and reduction in ATP synthesis, represent critical events.21 However, the roles of mitochondrial dysfunction in the graphene-mediated cytotoxicity of microglia are still unclear.

As highly dynamic organelles, mitochondria undergo continuous events, fusion and fission.22 These dynamic processes are required for determining mitochondrial morphology as well as diverse cellular functions, including cellular calcium homeostasis, metabolite synthesis, energy generation, and apoptosis.23 In mammalian cells, a family of protein involved in mitochondrial shape exists that regulate the balance between mitochondrial fusion and fission.24 Among them, dynamin-related GTPase, namely outer membrane mitofusins (Mfn-1 and Mfn-2), and inner membrane optic atrophy 1 (Opa1), control fusion. In contrast, dynamin-related protein 1 (Drp1) 4 ACS Paragon Plus Environment

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and fission protein 1 (Fis1) mediate mitochondrial fission.25 Abnormal mitochondrial dynamics has been recognized as a critical pathway leading to mitochondrial dysfunction and eventual cell death.26 Recently, considerable effort has been directed towards elucidating the roles of

mitochondria in graphene-induced toxicity.19,21

There have been recent reports suggested that surface properties of graphene play a crucial role in the structure-activity relationships that may influence its biological effects.10,21 However it is still unknown how the graphene functionalized with different functional groups affect the behaviors of microglia. Considering that mitochondrial dynamics has profound impacts on mitochondrial function, we developed a cell-based functional screening system tranfected with DsRed2-mito to study the effect of graphene oxides (GO), and polyethylene glycol (PEG)- and polyethylenimine (PEI)-functionalized GO on mitochondria dynamics in BV-2 cells. In this report, we demonstrated that positively charged GO-PEI promotes mitochondrial fragmentation with a concomitant increase in mitochondrial recruitment of Drp1. Moreover, GO-PEI treatment results in loss of ∆Ψm, increased mitochondrial ROS production, and reduction of ATP synthesis, which eventually contribute to apoptotic and autophagic cell death of microglia dependent on Drp1-mediated mitochondrial fission.

2. EXPERIMENTAL SECTION Preparation and Characterization of Graphene Derivatives. Graphene oxide (GO), polyethylene glycol (PEG)-functionalized GO (GO-PEG) were purchased from 5 ACS Paragon Plus Environment


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(Nanocs, Inc., Boston, MA). Preparation of polyethylenimine (PEI)-functionalized GO (GO-PEI) was performed according to the method as described.21 Briefly, GO suspension was diluted to ~1 mg/mL with deionized water, and a solution of 1 mg/mL PEI (MW 25,000, Sigma, St Louis, MO, USA) was then added to GO solution with constant stirring until a final GO/PEI weight ratio of 1:4 was obtained. After vortexing and sonication for 10 min, EDC was added and the mixture was stirred for an additional 24 h at room temperature. Finally, the resulting GO-PEI was precipitated by centrifugation at 13, 000 rpm for 2 h, and washed adequately with distilled water.

Atomic force microscopy (AFM; Bruker AXS, Berlin, Germany) analysis showed that the thickness of GO, GO-PEG, and GO-PEI was 1.1 nm, 5.8 nm, and 11.2 nm, respectively, with an average size ranging from 0.1 µm to 2.0 µm (Figure 1a, b). The spectra of fourier transform infrared spectroscopy (FT-IR; iN10 MX IR, Nicolet) confirmed the chemical structure of these graphene derivatives (Figure 1c). Zeta-potential (Malvern Nano ZS, Nalvern, UK) values of GO, GO-PEG, and GO-PEI were -18.5 mV, -6.4 mV and +34.2 mV in culture medium, respectively, consistent with previous reports indicating that GO and GO-PEG are negatively charged, whereas GO-PEI is positively charged.21 All graphene derivatives were homogeneously dispersion in culture medium, and were shown to be negative for endotoxins using the limulus amebocyte lysate test (Pyrogent 5000®; Lonza, Walkersville, MD).


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Figure 1. Characterization of graphene derivatives. (a) Typical AFM topography images of GO, GO-PEG, and GO-PEI. The size distribution of each graphene derivative similarly ranges from 0.1 µm to 2.0 µm. (b) Surface thicknesses of GO, GO-PEG, and GO-PEI. (*p < 0.05 vs. GO. n=5). (c) FT-IR spectra of GO, GO-PEG, and GO–PEI.

Cell Culture and DNA Transfection. BV-2 cell lines were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China), and the cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Life Technologies, Carlsbad, CA, USA), streptomycin (10 µg/ml) and penicillin (10 U/ml) (Invitrogen, Carlsbad, CA), and maintained at 37 °C in a humidified incubator with 5% CO2. To visualize mitochondria, BV-2 cells were transfected with DNA encoding 7 ACS Paragon Plus Environment


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pDsRed2-Mito (Clontech, Palo Alto, CA, USA) after reaching 70-80% confluence as described.27 This vector encodes red fluorescent protein derived from Dicosoma sp. fused to mitochondrial targeting sequence from subunit VIII of human cytochrome c oxidase.28 The DsRed2-mito expressing BV-2 cells were then selected for resistance to 4 µg/mL blasticidin (Invitrogen, Carlsbad, CA, USA). For the image-based screening of graphene derivatives, BV-2 cells expressing DsRed2-mito were grown in a 24-well plate, and then each graphene derivative at 50 µg/mL was added to each well. Morphological changes in the mitochondrial network were observed 6 h after incubation with each graphene derivative under a confocal laser scanning microscope (CLSM-710; Zeiss, Jena, Germany).

Analysis of Mitochondrial Morphology and Immunocytostaining. BV-2 cells transfected with DsRed2-mito were incubated with or without increasing concentrations of graphene derivatives for 6 h, then washed twice and fixed with 4% paraformaldehyde in PBS for 15 min at 37 °C, followed by staining with 0.5 µM MitoTracker green (Invitrogen, Carlsbad, CA, USA) for 15 min at 37 °C. Mitochondrial morphology was imaged

using a Zeiss LSM-710 confocal

microscope as described previously.29 To determine the localization of Drp1, BV-2 cells were seeded into 12-well chamber slides and then fixed with 4% paraformaldehyde for 20 min at 37 °C, permeabilized with 0.1% Triton X-100 for 20 min, and nonspecific binding sites was saturated by incubation with 2% bovine serum albumin (BSA) in PBS for 30 min at room temperature, followed by 1 h of incubation with anti-Drp1 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, 8 ACS Paragon Plus Environment

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USA) at a dilution of 1/100 at room temperature. After rinsing with PBS, the cells were incubated with goat anti-mouse Alexa Fluor 488 antibody at a dilution of 1/100 for 1 h at room temperature, co-stained with a blocking solution containing 0.5 µM Mitotraker green or 5 µg/mL DAPI. The Fluorescent images were obtained using a confocal microscope.

Cell Uptake Study and MTT Assay. The increase in cellular internalization of nanoparticles correlates with an increase in cellular granularity.30 Therefore, cellular uptake of graphene derivatives by BV-2 cells was quantified by determining the intensity of light side-scatter (SSC) via flow cytometry as described previously.31 For the cell viability assay, BV-2 cells were seeded on 96-well plates at a density of 5 × 104 per well. Twenty-four hours after plating, the cells were incubated with increasing doses of graphene derivatives (0-50 µg/mL) for 24 h and 48 h. MTT assay was then carried out to evaluate the effect of graphene derivatives on the viability of BV2 cells according to the manufacturer's instructions (Sigma, St. Louis, MO). In the assays using pan caspase inhibitor, 50 µM zVAD-fmk (R&D Systems, Minneapolis, MN) was added 1 h before the cells were exposed to GO-PEI. For the autophagy inhibition assay, BV-2 cells were pretreated with 5 mM 3-methyladenine (3-MA; Sigma, St Louis, MO, USA) for 1 h before GO-PEI treatment. For the mitochondrial fission assay, BV-2 cells were pretreated with 10 µM Mdivi-1, a selective inhibitor of Drp1 (Enzo Life Sciences, NY, USA) before GO-PEI treatment. Each condition was carried out in triplicate. Results for each treatment are presented relative to the control group. 9 ACS Paragon Plus Environment


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Determination of Mitochondrial Membrane Potential (∆Ψm) and Total Cellular ATP level.

The ∆Ψm in BV-2 cells was quantified using

5,5’,6,6’-tetrachloro-1,1’,3,3’- tetraethylbenzinidazolylcarbocyanine iodide (JC-1) dye fluorescent probe (Molecular Probes, Eugene, OR). JC-1 accumulates as aggregates in the mitochondria emitting red fluorescence when ∆Ψm depolarization, whereas the dye outflows into cytoplasm, JC-1 exists as monomers emitting red fluorescence. After treatment with different concentrations of GO-PEI (0-100 µg/mL) for 6 h, BV-2 cells were trypsinized and harvested, and then the fluorescence intensity of JC-1 was detected using a FACS can flow cytometry (BD Biosciences) after incubation with 5 µg/mL JC-1 for 30 min at room temperature in the dark. Intracellular ATP levels were measured with an ENLITEN ATP bioluminescence Detection Kit (Promega, Madison, WI) according to the manufacturer’s protocol. ROS Measurement. The levels of mitochondrial superoxide radicals were estimated using the MitoSOX Red fluorescent probe (Invitrogen, Carlsbad, CA). BV-2 cells were seeded into 96-well plates per well at a density of 5 × 104 cells, allowed to attach over night, followed by treatment of cells with GO-PEI (0-100 µg/mL) for 6 h in the absence or presence of either 0.5% (v/v) dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO, USA) or 20 mmol/L Mn(III) tetrakis (1-methyl-4-pyridyl) porphyrinpentachloride (MnTMPyP; Cayman Chemical, Ann Arbor, USA), a superoxide dismutase mimetic. Cells were then loaded with 2.5 mM MitoSOX Red incubated for 30 min at room temperature, and washed three times with PBS before fluorescence measurements. The intensity of MitoSOX Red 10 ACS Paragon Plus Environment

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fluorescence was quantified using a flow cytometry at an excitation wavelength of 510 nm and emission wavelength of 580 nm.

Drp1 Silencing. BV-2 cells were maintained at 37 °C in a humidified atmosphere of 5% CO2 until approximately 80% confluent in 24-well plate, and then the cells were transfected with either scrambled siRNA control (scram) or Drp1-siRNA (siDrp1) using a Lipofectamine RNAiMAX Transfection Reagent Kit (Life Technologies, Carlsbad, CA) according to the manufacturer’s protocol. A final siRNA concentration of 20 nmol/L was used in each well. The Drp1-siRNA duplex with the following

sense

and

antisense

5’-CTGGAGAGGAATGCTGAAA-3’, scrambled

siRNA

sequences

was

used:

5’-TTTCAGCATTCCTCTCCAG-3’.

(5’-CTGGAAATGGAGGGAACTA-3’;

sense)

The and

(5’-TAGTTCCCTCCATTTCCAG-3’; antisense) was used as a negative control. Knockdown of Drp1 was verified by western blot analysis 48 h after transfection.

Analysis of Apoptosis by Propidium Iodide (PI) Staining. BV-2 cells in the log-phase were seeded into 6-well culture plates at a density of 1 × 105 cells per well and maintained at 37 °C in a CO2 incubator in the absence or presence of GO-PEI. After treated with indicated concentrations of GO-PEI (0-50 µg/mL) for 24 and 48 h, the cells were then harvested, and fixed in 70% ethanol and stored at 4 °C overnight, and then centrifuged at 1,000 × g for 10 min. After washed with cold PBS three times, the cells were incubated with 50 µg/mL PI containing 10 µg/mL ribonuclease A for



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30 min at 37 °C. The percentage of cells in the sub-G1 phase of the cell cycle were then quantified by flow cytometry.

Protein Extract. BV-2 cells were washed twice with ice-cold PBS, trypsinized and harvested, then cell pellets were lysed on ice with cell lysis buffer consisting of 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate, 1 mM EDTA (Sigma, St. Louis, MO, USA), 1% protease inhibitor cocktail, and 1% phosphatase inhibitor cocktail (Roche Diagnostics Co., Indianapolis, IN). Cytoplasmic, nuclear, and mitochondrial fractions were isolated using a cytoplasmic extraction kit, NE-PER nuclear reagent kit, and mitochondria isolation kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s protocol, respectively. Protein concentration was determined by the bicinchoninic acid (BCA) assay method (Pierce, Rockford, IL).

Western Blot Analysis. Equal amounts of protein from each subcellular fraction were separated by 12% SDS–polyacrylamide gel electrophoresis (PAGE) and electrotransferred onto PVDF membranes (Millipore, Bedford, MA, USA). After blocking, the membranes were then probed with appropriate specific primary antibodies overnight at 4°C. Primary antibodies used for immunoblotting included anti-Drp1, anti-phosphorylated(p)-Drp1(Ser616), anti-p-Drp1(Ser637), anti-COX IV, anti-procaspase-3, anti-caspase-3, anti-PARP, anti-p62, anti-LC3, anti-PINK1 and anti-Parkin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-β-actin, anti-Mfn1, anti-Mfn2, anti-Fis1 (Sigma, St Louis, MO, USA), and anti-Opa1 (BD


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Biosciences). For protein detection, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. The primary antibodies were added at a dilution of 1/1000, and the secondary antibody was used at a dilution of 1/2000. The membranes were developed using an enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ, USA). Protein band density was quantified using Image J software (NIH, Bethesda, MD, USA) and expressed in arbitrary units. Autophagy Measurement. BV-2 cells (0.5 × 106 cells in 60 mm dish) were transfected with 0.5 µg of purified recombinant plasmid EGFP-LC3 using Lipofectamin™ 2000 (Invitrogen, Carlsbad, CA). EGFP-LC3 plasmid encoding a fusion protein consisting of enhanced green florescent protein (EGFP) and LC3 was obtained from Addgene (Cambridge, MA). Forty-eight hours later, cells are incubated in DMEM medium containing 500 µg/mL G418 (Invitrogen Life Technologies, Carlsbad, CA, USA) to select stable clones. Following treatment with increasing doses of GO-PEI (0-50 µg/mL) for 6 h, EGFP+ dots in the cells were observed under a CLSM and GFP-LC3 punctate spots were counted.LC3 positive cells were defined as cells that containing three or more EGFP+ dots.

Statistical Analysis. The data are presented as means ± standard deviation of three independent experiments. Comparisons between groups were analyzed by the t-test (two-sided) or One-way ANOVA. A p value less than 0.05 was considered statistically significant.

RESULTS 13 ACS Paragon Plus Environment


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Cell Uptake of Graphene Derivatives Depends on Surface Charge. Uptake potential is an important parameter for evaluating the toxicity mediated by nanoparticles in vitro.32 In the present study, three types of graphene derivatives, including GO, GO-PEG, and GO-PEI were used to address the effect of surface charge on their internalization by microglia. Cellular uptake of nanoparticles correlated well with the cellular granularity, which can be reflected by the intensity of SSC measured by flow cytometry.31,32 Figure 2a shows the time-dependent change of SSC intensity after treatment of BV-2 cells with each graphene derivative at 50 µg/mL. A time-dependent increase in SSC intensity but not FSC intensity was detected after exposed to graphene derivatives, which began efficiently as early as 3 h. Among them, GO-PEI was effectively taken up into cells compared to GO, while GO-PEG is less efficiently taken up by the cells. After 6 h of incubation, the amount of graphene derivatives endocytosis appeared to stop climbing and even was reduced slightly. Overall, BV-2 cells appeared to uptake the positively charged GO-PEI more than the negatively charged GO-PEG or GO (Figure 2a,b).


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Figure 2. Cellular uptake of graphene derivatives by microglia. (a) Cellular uptake of GO, GO-PEG, and GO-PEI at 50 µg/mL was quantified using side scattering (SSC) parameter detected by flow cytometry. (b) Typical flow cytometric SSC data was shown. Results are mean ± SD of three independent experiments. *p < 0.05, **p < 0.01 compared to GO group.

Effects

of

Graphene

Derivatives

on

Mitochondrial

Fragmentation.

Mitochondria are the central players in regulating cell survival and growth by integrating and modulating multiple cellular signaling pathways.33 To examine the effects of graphene derivatives on mitochondrial network, we used BV-2 cells with mitochondria labeled by DsRed2-mito as a cell-based functional screening system. As 15 ACS Paragon Plus Environment


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shown in Figure S1, Dsred2-mito shows no significant effect on cell viability of BV-2 cells after 48 h of transfection. CLSM images showed that the expression of DsRed2-mito exhibited an apparently filamentous pattern characteristic of mitochondria (Figure 3a). To reaffirm whether DsRed2-mito protein colocalizes with mitochondria,

BV-2

cells

were

co-loaded

with

MitoTracker

green,

a

mitochondria-specific dye. Superimposition of the two images revealed a high degree of overlap between DsRed2-mito staining and the mitochondrial staining (Figure 3a). As shown in Figure 3b, positively charged GO-PEI caused mitochondrial fragmentation dramatically as compared with control cells, where mitochondria exist as an elongate and form a mesh of highly interconnected filaments. The percentage of cells with fragmented mitochondria in GO-PEI-treated BV-2 cells was 38.2% compared to 15.1% of the control cells (Figure 3c). However, there is no significant mitochondrial fragmentation observed either in negatively charged GO- or GO-PEG-treated cells. Previous studies showed that mitochondria fission in microglia may be necessary for excessive production of proinflammatory mediators.28 Figure S5 shows the effects of graphene derivatives on the release of proinflammatory mediators. As expected, GO-PEI was found to elicit the release of tumor necrosis factor-α (TNF-α) and nitric oxide (NO). Since GO-PEI shows the potential to induce mitochondrial fission, we focused on defining the molecular mechanisms of its action and downstream cellular events.


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Figure 3. (a) BV-2 cells expressing DsRed2-mito were analyzed under a CLSM after staining with MitoTracker green. (b) Representative confocal images of morphological changes in mitochondrial network after treatment of cells with each graphene derivative. BV-2 cells tranfected with DsRed2-mito were exposed to GO, GO-PEG, and GO–PEI at 50 µg/mL for 6 h, respectively, and then imaged using a 17 ACS Paragon Plus Environment


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CLSM. Representative images are shown. (c) Quantitative analysis of mitochondrial morphology using ImageJ software. In each experiment, at least 20 cells were analyzed per condition. *p < 0.05 compared to the control group.

Effects of Graphene Derivatives on Expression of Mitochondrial Fission/fusion Proteins. To examine whether the mitochondrial fission machinery was involved in GO-PEI-induced mitochondrial fragmentation, the levels of mitochondrial fission and fusion proteins were analyzed. In agreement with the significant increase in mitochondrial fragmentation observed in GO-PEI-treated BV-2 cells, western blotting analysis of subcellular fraction revealed that GO-PEI increases Drp1 recruitment to mitochondria in a dose-dependent manner 6 h after incubation with GO-PEI (Figure 4a,b), which was confirmed by immunofluorescence study (Figure 4c) and quantitative colocalization analysis (Figure 4d). However, BV-2 cell treated with either GO or GO-PEG did not show any alternation in mitochondrial recruitment of Drp1 (data not shown). In addition, to reveal the potential effect of positive charge on graphene on mitochondrial fragmentation in BV-2 cells, we compared another positively charged amine-modified graphene (GO-NH2) (Figure S2, Table S1), and other nanomaterials with different physicochemcial characteristics from graphene, such as PEI-modified multiwall carbon nanotubes (MWCNTs-PEI) (Figure S3, Table S1) or gold nanoparticles (AuNPs-PEI) (Figure S4, Table S1). As shown in Figure S6, in contrast to MWCNTs-PEI or AuNPs-PEI, treatment with GO-NH2 also trigged a


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significant increase in mitochondrial recruitment of Drp1 in BV-2 cells, indicating that the positive charge on graphene could contribute to mitochondrial fragmentation.

Dephosphorylation of Drp1 at Ser637 permits mitochondrial translocation of Drp1, an obligatory step in the initiation of fission.34 Western blotting analysis further demonstrated that GO-PEI induced a significant decrease in the phosphorylation of Drp1 at Ser637 in BV-2 cells (Figure 4e,f). In contrast, another phosphorylation site of Drp1 (Ser616), which promotes mitochondrial fission, was significantly increased in cells treated with GO-PEI (Figure 4e,f). However, GO-PEI treatment did not caused any significant changes in other proteins involving mitochondrial fission and fusion, such as Fis1, Mfn1, Mfn2 and Opa1 (Figure 4e,f). These results demonstrated that Drp1 dephosphorylation at Ser637, which coordinates with Drp1 phosphorylation at Ser616, triggered mitochondrial recruitment of Drp1, thus resulting in mitochondrial fragmentation in GO-PEI-treated BV-2 cells.



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Figure 4. (a) BV-2 cells were stimulated with GO–PEI at 25, 50, 100 µg/mL for 6 h, and the cytoplasmic and mitochondrial fractions were prepared to analyze the translocation of Drp1 to mitochondria by western blotting. COX IV and β-actin were served as mitochondria and cytoplasmic markers, respectively. (b) The ratio of Drp1 expression in mitochondria and cytoplasm were performed by densitometric analysis. Results are mean ± SD of three independent experiments. *p < 0.05, **p < 0.01 compared to control group. (c) Recruitment of Drp1 into mitochondria 6 h after treatment with 50 µg/mL GO-PEI. The yellow color in the merged image represents an apparent colocalization of Drp1 (green) with DsRed2 (red) after treatment with GO-PEI. (d) Colocalization analysis of Drp1 with mitochondria obtained by the quantitative analysis of CLSM images shown in (c). *p < 0.05 compared to the control group. (e) Western blotting analysis of mitochondrial fission/fusion proteins in BV-2 cells 6 h after incubation with GO, GO-PEG, and GO-PEI (50 µg/mL), respectively. (f) The relative levels in the expression of mitochondrial fission/fusion protein were performed by densitometric analysis. Results are mean ± SD of three independent experiments.*p < 0.05 vs. control group.

Positively Charged GO-PEI Leads to Mitochondrial Dysfunction and ROS Generation. To explore whether GO-PEI-mediated mitochondrial fragmentation contributes to mitochondrial dysfunction, ∆ψm and total cellular ATP levels were determined. Compared with untreated BV-2 cells, GO-PEI treatment resulted in a



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significant collapse of ∆ψm after 6 h of incubation in a dose-dependent manner using a unique fluorescent cationic dye JC-1(Figure 5a,b). ∆ψm is essential in the stabilization and maintenance of mitochondrial function, which is critical for ATP production. Next, we assayed total cellular ATP levels in BV-2 cells treated with GO-PEI. As indicated in Figure 5c, GO-PEI treatment also caused a decrease in the level of cellular ATP levels with a similar pattern as that of ∆ψm. To examine the contribution of ROS induced by GO-PEI to mitochondrial damage, intracellular superoxide production was measured using MitoSOX Red that selectively target mitochondria. As shown in Figure 5d and e, treatment with GO-PEI for 6 h resulted in an increase in fluorescence intensity in dose-dependent manner. However, pretreatment of BV-2 cells with the radical scavenger dimethyl sulfoxide (DMSO, 0.5%) for 30 min totally prevented the induction of superoxide production (Figure 5f), confirming that mitochondrial ROS was induced by GO-PEI. To check the specificity of MitoSOX assay in detecting superoxide production, BV-2 cells were further pretreated with MnTMPyP, a superoxide dismutase mimetic, at the dose of 20 mmol/L. Similar to the data with DMSO, treatment with MnTMPyP effectively quenched the increase in MitoSOX fluorescence intensity following GO-PEI treatment (Figure 5f).


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Figure 5. GO-PEI triggered ∆ψm depolarization, ATP deleption, and production of mitochondrial ROS. BV-2 cells were treated with GO-PEI (25, 50, 100 µg/mL) for 6 h, and then (a) flow cytometric analysis of changes in ∆ψm using JC-1 dye. Q2: red 23 ACS Paragon Plus Environment


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fluorescent JC-1 aggregates; Q4: green fluorescent JC-1 monomers. (b) Quantitative data confirm that GO-PEI induces ∆ψm loss in BV-2. (C) Total cellular ATP levels assays were carried out using an ATP bioluminescence assay kit. (d) BV-2 cells were treated with increasing dose of GO-PEI (25, 50, 100 µg/mL) for 6 h, followed by incubation with MitoSOX Red, and then observed under a CLSM. Representative images are shown. (e) Quantitative fluorescence intensity was analyzed by ImageJ software. The mitochondrial ROS level was increased by GO-PEI. (f) Effects of radical scavengers on GO-PEI-induced ROS generation. Results are expressed as means ± SD of at least three independent experiments. *p < 0.05, *p < 0.01 compared to the control group.

Positively Charged GO-PEI Induces Apoptotic Cell Death. Since mitochondrial fragmentation has been implicated in apoptotic cell death and precedes activation of effector or initiator caspase,35 the above findings prompted us to identify the mode of cell death induced by GO-PEI. As indicated in Figure 6a, treatment with GO-PEI at concentrations higher than 50 µg/mL for 24 and 48 h resulted in a significant reduction in cell viability of BV-2 cells compared to the untreated-cells. Cell cycle analysis with PI staining showed that the number of apoptotic cell death increased by 37.4 % and 41.5% 24 h after treatment with 25 µg/mL and 50 µg/mL of GO-PEI, respectively (Figure 6b). Western blotting analysis revealed that GO-PEI induced a significant increase in the expression of cleaved caspase-3 and PARP, indicating that the cytotoxicity elicited by GO-PEI may manifest as apoptotic cell death (Figure 6c). 24 ACS Paragon Plus Environment

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However, pretreatment with 20 µM of zVAD-fmk, a pan-caspase inhibitor, did not totally suppress GO-PEI-induced apoptotic cell death of BV-2 cells (Figure 6d).

Figure 6. (a) Cell viability of BV-2 cells after GO-PEI treatment. BV-2 cells were treated with increasing doses of GO-PEI for 24 h and 48 h, respectively, and MTT assays was performed to determine cell viability of BV-2 cells. Results are presented as the mean ± SD of three independent experiments.*p < 0.05 compared to the control group. (b) BV-2 cells were treated with 25-50 µg/mL GO-PEI for 24 h, followed by apoptosis assay using flow cytometry with PI staining. (c) Western blot analysis of caspase-3 and cleaved-PARP in GO-PEI-treated BV-2 cells.. (d) The relative levels of caspase-3 and cleaved PARP were performed by densitometric analysis. *p < 0.05.



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GO-PEI Induces Autophagic Cell Death in BV-2 Cells. Autophagy, which is independent of the apoptotic pathway, has been considered as a novel type of cell death, and various nanoparticles have been shown to possess the potential to induce autophagy.36 To inversitgate whether GO-PEI triggers autophagy in BV-2 cells, the expression patterns of LC3-II and cytoplasmic p62 were anayzed after treatment with GO-PEI. Immunofluorescence assay showed that punctuated LC3, the hallmark of autophagosome formation, was apparently detected in BV-2 cells treated with 50 µg/mL of GO-PEI for 24 h (Figure 7a,b). Cells were also treated for the same time with rapamycin (20 nM), as the positive controls for induction of autophagy. To further detect lysosomal localization of LC3 puncta, we examined the colocalization of LC3 with lysosome using LysoTracker-red, a fluorescent lysosomal marker. As expected, GO-PEI treatments highly led to an increased colocalization of LC3 puncta (green) with LysoTracker-red in the cytoplasm of BV-2 cells (Figure 7b). As shown in Figure 7c,d, western blot analysis demonstrated that GO-PEI clearly increased the conversion of LC3-I into LC3-II, and decreased the protein level of p62, indicating autophagy activation in BV-2 cells. Next, we investigated whether GO-PEI stimulates the expression of mitophagy-related proteins. Figures 7e,f shows that both PINK1 and Parkin accumulate in mitochondrial fractions of GO-PEI-treated BV-2 cells, indicating the involvement of mitophagic pathway in the clearance of mitochondrial fragmentation by GO-PEI. To further confirm GO-PEI-induced autophagy, BV-2 was treated with GO-PEI alone or along with 3-MA, an inhibitor of autophagy. As shown in Figure 7g,h, the levels of LC3-II and p62 expression by GO-PEI were abolished 26 ACS Paragon Plus Environment

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when 3-MA was added, suggesting that autophagic cell death may contribute to decreased cell viability in GO-PEI-treated BV-2 cells.



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Figure 7. GO-PEI induces autophagic cell death in BV-2 cells. (a) BV-2 cells were treated with 25-50 µg/mL GO-PEI for 24 h, and GFP-LC3 labeled autophagosomes per cell (n = 20) were counted and shown graphically. *p < 0.05 compared to the control group. Cells treated with rapamycin (20 nM) were served as the positive controls for induction of autophagy. (b) Immunocytostaining for the colocalization of GFP-LC3 (green) with Lysotracker (red) after GO-PEI (50 µg/mL) for 24 h in BV-2 cells. Autophagosomes are recognized as punctate structures. (c) Immunoblotting analysis of the conversion of LC3-I to LC3-II and degradation of p62. (d) The relative intensity of LC3-II and p62 were quantified by densitometric analysis. (e,f) The mitochondrial fractions were isolated from BV-2 cells treated with or without GO-PEI, and then analyzed by western blotting with antibodies specific for the indicated proteins. The relative intensity of PINK1 and Parkin was quantified by densitometric analysis. (g,h) GO-PEI-induced autophagy is abrogated by autophagy inhibitors. Western blot analysis of LC3-II and degradation of p62 in BV-2 cells treated with 50 µg/mL of GO-PEI for 24 h in the presence of 3-MA. The relative intensity of LC3-II


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and p62 were calculated by densitometric analysis. *p

Drp1-Dependent Mitochondrial Fission Mediates Toxicity of Positively

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