Biol Trace Elem Res (2014) 159:393–398 DOI 10.1007/s12011-014-0027-3

Distribution of Graphene Oxide and TiO2-Graphene Oxide Composite in A549 Cells Chan Jin & Fude Wang & Ying Tang & Xiangzhi Zhang & Jianqiang Wang & Yongji Yang

Received: 10 April 2014 / Accepted: 22 May 2014 / Published online: 30 May 2014 # Springer Science+Business Media New York 2014

Abstract Graphene and its derivatives are increasingly applied in nanoelectronics, biosensing, drug delivery, and biomedical applications. However, the information about its cytotoxicity remains limited. Herein, the distribution and cytotoxicity of graphene oxide (GO) and TiO2-graphene oxide composite (TiO2-GO composite) were evaluated in A549 cells. Cell viability and cell ultrastructure were measured. Our results indicated that GO could enter A549 cells and located in the cytoplasm and nucleus without causing any cell damage. TiO2 nanoparticles and GO would be separated after TiO2-GO composite entered A549 cells. TiO2-GO composite could induce cytotoxicity similar to TiO2 nanoparticles, which was probably attributed to oxidative stress. These results should be considered in the development of biological applications of GO and TiO2-GO composite. Keywords Graphene oxide . TiO2-graphene oxide composite . A549 cells . Cytotoxicity

Introduction Graphene is a two-dimensional material that exhibits outstanding mechanical, thermal, optical, and electronic properties and has been widely applied in nanoelectronics, catalysis, biosensing, drug delivery, and biomedical applications [1–5]. C. Jin (*) : F. Wang : X. Zhang : J. Wang Shanghai Institute of Applied Physics, Chinese Academy of Sciences, 201800 Shanghai, China e-mail: [email protected] Y. Tang : Y. Yang Institute of Biophysics, Second Military Medical University, 200433 Shanghai, China Y. Yang e-mail: [email protected]

Graphene and graphene oxide (GO) have been actively investigated to build new composite materials [6]. TiO2-GO composite provides enhanced degradation of methylene blue and bacteria [7]. Given the broad applications of graphene and its derivatives, it is important to evaluate their potential risks to human health. It has been reported that graphene and GO are highly stable under physiological conditions and show excellent biocompatibility with various cell types and living systems [8, 9]. However, recent studies demonstrated that concentration-dependent cytotoxicity during a 24-h exposure to GO was due to the generation of reactive oxygen species and indicated that nonfunctionalized GO could kill bacteria and cause cell membrane damage [10]. And, till now, there is a lack of such information for newly developed graphene derivatives such as TiO2-GO composite. In the present study, the distribution and cytotoxicity of GO and TiO2-GO composite were evaluated on the A549 human lung adenocarcinoma cell line. The distribution of GO and TiO2-GO composite was detected by transmission electron microscopy (TEM). Cell viability was detected by cell counting kit-8 (CCK-8) assay. Furthermore, the nearestneighbor structure of Ti in a single cell was detected based on the absorption spectrum.

Materials and Methods Preparation and Characterization GO was synthesized by a modified Hummers’ method [11, 12]. TiO2-GO composite was obtained via a hydrothermal method. Briefly, 0.5 mg GO was dissolved in 20 ml C2H5OH/H2O and ultrasonicated for 3 h. Then, 300 mg anatase TiO2 (25 nm; purchased from Aladdin Industrial Corporation, China) was added to the GO solution to create a homogeneous suspension. The suspension was then placed

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in a Teflon-sealed autoclave and maintained at 150 °C for 12 h to achieve the deposition of TiO2 on the GO. Finally, the composite was filtered, washed with deionized water three times, and dried at room temperature. Transmission electron microscopy and Raman spectroscopy were applied to determine the characteristics of GO, TiO 2 , and TiO 2 -GO composite. Cell Culture The A549 cell line, a human lung adenocarcinoma derived by explant culture from the peripheral airways of a Caucasian male with lung cancer, was established in 1972 and had characteristic features of type II cells of the pulmonary epithelium, including lamellar bodies [13]. This cell line has been a popular model extensively referenced in toxicology literatures [14–16]. The A549 cells were purchased from the China Center for Type Culture Collection (Wuhan, China). A549 cells were cultured in MEM (Gibco BRL, Invitrogen AG, Basel, Switzerland) supplemented with 10 % fetal bovine serum (Sijiqing Biological Engineering Materials Co., Ltd., Hangzhou, China) and 1 % penicillin/streptomycin. Cells were maintained at 37 °C in a humidified 5 % CO 2 atmosphere. Cell Viability Cell viability was evaluated by CCK-8 (Beyotime Institute of Biotechnology, Jiangsu, China) according to the manufacturer’s instruction. Briefly, A549 cells were seeded in 96well culture plates with 5×103 cells in 100 μl DMEM per well; after 24 h of cell attachment, A549 cells were treated with different concentrations (100 and 300 μg/ml [17]) of GO, TiO2, and TiO2-GO composite for 4 h. Then, 10 μl 2-(2methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8) solution was added to each well, and the cells were further incubated for 1 h in the incubator. Cells without any treatment were served as the control. The optical density (OD) of each well at 450 nm was recorded by a microplate reader (Thermo Scientific Varioskan Flash, Thermo Fisher Scientific Inc., USA). Cell Ultrastructure After treatment with GO, TiO2, and TiO2-GO composite for 4 h, A549 cells were fixed with 2.5 % glutaraldehyde and postfixed with 1 % OsO4 [18]. Cells were then dehydrated through a graded series of ethanol and acetone and embedded in Epon 812 [19–21]. Ultrathin sections (60 nm) were cut using an ultramicrotome (UC6, Leica Ltd. Co, Germany) and transferred onto copper mesh grids, stained with uranyl acetate

Jin et al.

and lead citrate and observed by TEM (H-7650, Hitachi, Japan). Structure of Ti in Cells The X-ray absorption near-edge structure (XANES) data of Ti K-edge were collected at BL14W1 (Shanghai Synchrotron Radiation Facility, Shanghai, China). Higher harmonics were reduced sufficiently by detuning the double-crystal Si(111) monochromator. XANES experiments were conducted in transmission detection mode for TiO2 and TiO2-GO composite and fluorescence detection mode for cell samples treated with TiO2 and TiO2-GO composite. Energy calibration was performed using a Ti foil. The raw data were normalized by the IFEFFIT program [22, 23]. Distribution of Ti in a Single Cell The distribution map of Ti was collected by scanning transmission X-ray microscopy (STXM) with 3.5 GeV electron energy and 160–210 mA ring current at BL08U1 (Shanghai Synchrotron Radiation Facility, Shanghai, China). Two photon energies, E1 =461.5 eV and E2 =463.1 eV, which were just below and above the L2-edge absorption energy of Ti were chosen for detection. The distribution of Ti in a single cell was calculated by subtraction and ratio methods. The image and step size were 35 μm×35 μm and 50 nm, respectively. Statistical Analysis All numerical data were presented as mean ± standard deviation (S.D.) of three separate experiments and analyzed by a one-way ANOVA followed by Dunnett’s t test for comparisons between groups, using statistical program SPSS (SPSS Inc., USA). Asterisk denoted statistical significance (*

Distribution of graphene oxide and TiO2-graphene oxide composite in A549 cells.

Graphene and its derivatives are increasingly applied in nanoelectronics, biosensing, drug delivery, and biomedical applications. However, the informa...
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