In Vitro Pulmonary Toxicity of Reduced Graphene Oxide-Nano Zero

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In Vitro Pulmonary toxicity of Reduced Graphene Oxide-Nano Zero Valent Iron Nanohybrids and Comparison with Parent Nanomaterial Attributes Qixin Wang, Arvid Masud, Nirupam Aich, and Yun Wu ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b02004 • Publication Date (Web): 25 Aug 2018 Downloaded from http://pubs.acs.org on August 28, 2018

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ACS Sustainable Chemistry & Engineering

In Vitro Pulmonary Toxicity of Reduced Graphene Oxide-Nano Zero Valent Iron Nanohybrids and Comparison with Parent Nanomaterial Attributes by Qixin Wang1, §, Arvid Masud2, §, Nirupam Aich2,*, Yun Wu1,* 1

Department of Biomedical Engineering, School of Engineering and Applied Sciences, University at Buffalo, The State University of New York, Buffalo, N.Y. 14260 2 Department of Civil, Structural and Environmental Engineering, School of Engineering and Applied Sciences, University at Buffalo, The State University of New York, Buffalo, N.Y. 14260 Invited Submission to ACS Sustainable Chemistry & Engineering Sustainable Nanotechnology Organization (SNO) Conference 2017 Virtual Special Issue Date: August 6, 2018 §

Equally Contributing Authors

* Corresponding Authors Nirupam Aich, Ph.D. Department of Civil, Structural and Environmental Engineering University at Buffalo, The State University of New York 232 Jarvis Hall Buffalo, NY, 14260 Phone: 716-645-0977 Email: [email protected] Fax: 716-645-3733 Yun Wu, PhD Department of Biomedical Engineering University at Buffalo, The State University of New York 215D Bonner Hall Buffalo, NY 14260 Email: [email protected] Phone: 716-645-8498 Fax:716-645-2207

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ABSTRACT Conjugation of individual nanomaterials to form ‘nanohybrids’ has been the recent focus of advanced material synthesis with the aim to achieve enhanced properties and synergistic functionalities. However, nanohybrids may induce uncertain and unknown biological interactions and toxicological responses that are likely to be unique and altered from their component nanomaterial attributes. In this study, reduced graphene oxide-nanoscale zero valent iron (rGO-nZVI), a multifunctional nanohybrid with promise for environmental remediation, has been systematically evaluated for in vitro toxicity to human bronchial epithelial cells (BEAS-2B). Careful synthesis of rGO-nZVI was performed by chemical co-reduction of graphene oxide (GO) and iron salt precursors, followed by evaluation of their physicochemical properties and colloidal stability in the biological media. A comprehensive assessment of biological interactions and toxicological outcomes of rGO-nZVI and its parent materials i.e., GO, rGO, and nZVI on BEAS-2B cells included cellular uptake, cell viability, cell membrane integrity, reactive oxygen species (ROS) generation, and cell cycle analyses. The toxic behavior of rGO-nZVI nanohybrids were found to be in between that of rGO/GO (most toxic) and nZVI (least toxic); however, majorly governed by rGO/GO toxicity and its mechanisms. This study sheds light on the importance of sustainable design strategies for the next-generation complex and hierarchical nanostructures. Keywords: Safer-by-design, iron nanoparticles, nanotoxicity, nanocomposite, pulmonary toxicity.

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INTRODUCTION Engineered nanomaterials (ENMs), both carbon based (e.g., fullerene, carbon nanotubes, graphene) and metallic (nano zero valent iron or nZVI or Fe0, titanium dioxide or TiO2, silver or Ag), have been used in wide range of applications including electrical and electronics, construction and aerospace, industrial and agricultural, biomedical, energy, and environmental fields. On the other hand, many ENMs have been identified as emerging environmental contaminants with the potential to emanate harmful human health hazards including pulmonary toxic effects upon inhalation exposure.1 Nanotoxicity research in the past decades has been restricted mostly to low dimensional singular ENMs and identified relationship of toxicity with ENMs’ physicochemical properties e.g., size (diameter or length), shape (sphere vs rods), surface chemistry and coating, crystallinity, electronic structures.2-3 Recently material synthesis in nanoscale has proceeded towards more unique and complex nanostructures.

A new class of ENM, nanohybrids (NHs), has appeared with the aim to

achieve enhanced- or multi-functionalities.2-5 NHs are conjugated forms of two or more pre-synthesized ENMs which act as a single unit and manifest enhanced and unique emergent properties that are not equal to the sum of the parts. For example, nZVI nanoparticles are widely used metal ENMs for environmental remediation due to their redox and magnetic properties.6 While deposited on highly conductive 1D carbon nanotubes (CNTs) or 2D reduced graphene oxide (rGO) nanosheets, nZVI particles become well-dispersed and smaller in size, have improved reactivity and better contaminant removal capacity.7 Such multifunctional and ever-expanding class of NHs with complex structures, composition, and altered properties are yet to be studied for their environmental health and safety (EHS) including their inhalation exposure to humans and resulting toxicity.2-5 Hybridization of two or more ENMs can lead to alterations and emergence of unique properties including electronic band structure, reactivity, dissolution (for metals), and

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dimensionality.2-5 These changes can lead to unprecedented alterations in their biological interactions and potential adverse health outcome. To address the uncertainty regarding the environmental and human health risks of NHs, we selected rGO-nZVI NH as the model NH. nZVI is the most used engineered nanomaterial for in-situ soil and groundwater remediation. The high redox activity and adsorptive capacity enable nZVI to remove heavy metal and organic contaminants. nZVI has been applied to 77 pilot and field scale polluted sites worldwide8-9 and also in industrial wastewater treatment plants.10 However, nZVI particles agglomerate due to van der Waals and magnetic attraction forces, decreasing reactivity and adsorption. To overcome these challenges, co-reduction of iron salts and GO have been performed to obtain rGO-nZVI NHs because GO provide large accessible specific surface area, good mechanical durability, excellent electron transfer, and finally improved contaminant removal ability.11 The promising nature of nZVI and rGO-nZVI for environmental applications raises concerns regarding their adverse human and environmental health effects.12 Inhalation of nZVI and related composites has been identified as the major route of exposure during their manufacturing and deployment stages - raising concerns about their adverse pulmonary health effects including chronic obstructive pulmonary disease (COPD). The limited research has shown that nZVIs cause reactive oxygen species (ROS) generation, oxidative stress, and cytotoxicity at low dosage (10 µg/mL) when exposed to airway epithelial cells.12-13 Yet, due to hybridization of nZVIs with rGO, these interactions will be further complicated and will remain uncertain. Few recent studies have reported toxicity induced by GO-based NHs. For example, Khan et al. showed that GO-Ag NH induced toxicity in A549 lung alveolar epithelial cells and reduced the cell viability with a dose-dependent manner; however, the contributions from and comparisons with the individual parent materials were not studied.14 Jin et al. reported that GO-TiO2 NH and TiO2 nanoparticles showed toxicity and oxidative stress; however, the

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study didn’t show any dose-dependent toxicity measurement.15 Thus, both studies lack providing a complete mechanistic understanding of the NH cytotoxicity, and also fail to address the critical knowledge gap: how and to what extent the hybridization and contributions from the parent materials impacts the NH toxicity. Moreover, the cytotoxicity of rGO-nZVI NH has not yet been studied and needs to be addressed given its promises in diverse applications. The present study aims to systematically evaluate and compared the toxicity of rGO-nZVI NH with its parent materials. To achieve this, we synthesized rGO-nZVI NH by chemical co-reduction of GO and iron salt precursor, and compared rGO-nZVI NH toxicity with the toxicity induced by GO, rGO, and nZVI. We characterized the physicochemical properties and colloidal stability of the rGO-nZVI NH and parent materials using transmission electron microscopy, X-ray diffraction spectroscopy, Raman spectroscopy, and time resolved dynamic light scattering. We exposed the NHs and component materials to human bronchial epithelial cells (BEAS-2B) to evaluate their in vitro toxicity. We elucidated the toxicity mechanisms of rGO-nZVI NH, and investigated the roles of hybridization and parent materials in NH toxicity.

MATERIALS AND METHODS Materials.

1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was purchased from

Avanti Polar Lipids (Alabaster, AL). Bovine serum albumin (BSA) and D-glucose were purchased from Fisher Scientific (Fair Lawn, NJ). Roswell Park Memorial Institute medium or RPMI 1640 medium, fetal bovine serum (FBS), penicillin-streptomycin, trypan blue, LDH Cytotoxicity Assay Kit, H2DCFDA, RNase A, and propidium iodide were purchased from Thermo Fisher Scientific (Carlsbad, CA).

Single layer graphene oxide (GO, >99.3 wt%,

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thickness 0.43-1.23 nm) and zero valent iron nanopowder (Fe0, 99.5%, 25 nm, partially passivated) were purchased from US Research Nanomaterials, Inc. (Houston, TX). Sodium Borohydride (NaBH4) and ferric chloride hexahydrate (FeCl3.6H2O) were purchased from Fisher Scientific (Fair Lawn, NJ) and Acros Organics (Geel, Belgium) respectively.

rGO-nZVI NH Synthesis.

rGO-nZVI NH was synthesized following a well-established wet

chemistry method.16 Briefly, GO (100 mg) was dispersed in DI water (200 mL) by sonication for 2 hours with a microtip (1/16”) based ultrasonic dismembrator (Q700, Qsonica Sonicators, Newtown, CT), and then 1 g of FeCl3.6H2O was added to the dispersion. For co-reducing the iron salt and GO, 20 mL of NaBH4 solution (1.65 M) was added drop-wise (1 drop/second) into the mixture with a peristaltic pump. The reaction was carried out in an inert atmosphere with N2 gas flow (0.3 SLPM) under continuous magnetic stirring (300 rpm) at room temperature. After adding the NaBH4, the suspension was kept stirring for another half an hour under N2 gas. The solution was washed with ethanol and DI water for couple of times through vacuum filtration (0.45 µm high volume low pressure (HVLP) filter) for removing excess ions. The products were dried overnight in oven at 60 oC. The dried samples were autoclaved before using it for further experiments. The mass content of rGO and nZVI in the dried samples were found to be 77% and 23%; these values were determined experimentally by performing inductively coupled plasma optical emission spectroscopy (ICP-OES) details of which is provided in the supporting information section S1. Although, the commercial zero valent iron nanopowder, mentioned above and termed as nZVI throughout the rest of the manuscript, was used to elucidate the toxicological behavior

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of nZVI; we also synthesized a batch of nZVI via reduction of FeCl3.6H2O using NaBH4 following the same method described above except for the addition of GO in the mixture. The toxicological effects of this lab synthesized nZVI was compared with that of commercial nZVI to validate the use of commercial nZVI for this study. Similarly, since GO and rGO had shown differential toxicity behavior in the previous literature, rGO was synthesized by reduction of GO using NaBH4 in the absence of iron-salt precursor to evaluate and compare its toxicity with that of rGO-nZVI NH.

Physicochemical property and colloidal stability characterization.

High resolution

transmission electron microscopy (HRTEM, JEOL JEM 2010, JEOL USA, Inc.) was performed at an accelerating voltage of 200 KV to image the physical morphology of the GO, nZVI, and rGO-nZVI NHs. One mg of sample was sonicated in 10 mL ethanol with ultrasonic dismembrator for HRTEM sample preparation. One drop of the dispersion was placed on a carbon-coated copper grid (100 mesh, Ted Pella, Inc., Redding, CA) and dried for 1 h before analysis. The X-ray diffraction (XRD) analysis of the samples was performed at room temperature by Rigaku Ultima IV (Rigaku Corporation) using the Kα emission of a Cu X-ray source (λ = 1.5418 Å). The incident angle range was kept in the range from 5° to 90° with a scan rate of 2º/min and step size of 0.02º. The vibrational modes and lattice order of the molecular structures were studied using Raman Microscope (Renishaw InVia, Renishaw plc.) with incident laser wavelength of 514

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nm. One mg of dried sample was placed as a thin layer on a glass slide to carry out the Raman spectroscopy. Time resolved dynamic light scattering (TR-DLS) was used to characterize the aggregation behavior of GO, rGO, nZVI, and rGO-nZVI NHs in the toxicological media. The nanomaterials were placed in a dispersion medium which mimics the lung fluid and is a Ca2+/Mg2+ free phosphate buffer saline (PBS) solution containing 5.5 mM D-glucose, 0.6mg/mL

bovine

serum

albumin

(BSA),

and

0.01mg/mL

1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, lung surfactant) at pH=7.4.17 The mixture was sonicated using a microtip (1/16”) based ultrasonic dismembrator and had a concentration of 2 mg/mL in 10 mL dispersion medium. 20 µL of the dispersion was mixed with 8 mL of culture medium containing RPMI1640 medium supplemented with 10% FBS and 1x penicillin-streptomycin and vortexed for 2 minutes to achieve the final concentration of 5 µg/mL. One mL of the dispersion was placed in a standard quartz cuvette cell inside a Malvern ZetaSizer NanoZS (Malvern instruments, Inc., Westborough, MA) equipped with a 4 mW 633 m He-Ne laser to perform TR-DLS and continuous scattering data was collected at 15 s intervals for 1 h. The scattering data was converted by the Contin algorithm to obtain the hydrodynamic diameter of the ENMs and NH at different time intervals. To better understand the effect of the dispersion medium on the colloidal stability of the nanomaterials, we also performed TR-DLS of the similar samples in DI water prepared at the same concentration and by the same procedure. Moreover, we performed sedimentation test (detailed procedure provided in the supporting information section S2) of these nanomaterials in the dispersion media.

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Dispersion preparation for in vitro toxicity studies.

GO, rGO, nZVI, and rGO-nZVI NH

powders were first sterilized by autoclave. Then they were suspended in the dispersion medium to reach a concentration of 2 mg/mL. The suspensions were sonicated in ice-bath for 20 min (1 min on, 10 s off) with ultrasonic dismembrator before using for cellular uptake and cell viability tests.

BEAS-2B cell culture.

BEAS-2B normal human bronchial epithelial cells were purchased

from American Type Culture Collection (ATCC, Manassas, VA). BEAS-2B cells were cultured in RPMI1640 medium supplemented with 10% FBS and 1x penicillin-streptomycin. BEAS-2B cells were sub-cultured every 2 days.

Cellular uptake of the nanohybrid and its components.

The cellular uptake of GO, rGO,

nZVI, and rGO-nZVI NH was measured following an established protocol.18 Briefly, BEAS-2B cells were seeded in 12-well plates at 7.5×104 cells per well and allowed to grow overnight. The cells were treated with GO, rGO, nZVI, and rGO-nZVI NH individually at the concentration of 5 µg/mL. At 1, 2, 4, 8, 12, 24 and 48 h post-treatment, the nanomaterials were removed by washing cells with PBS twice. Then 200 µL 0.2 M NaOH was added to the cells and incubated at room temperature for 2 h to completely lyse the cells. Next, 50 µL dimethyl sulfoxide (DMSO) was added to the cell lysate. The concentration of GO, rGO, nZVI, and rGO-nZVI NH in the cell lysate was determined using optical absorbance

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spectroscopy at 525 nm. The cellular uptake of each sample was calculated using the following equation:

cellular uptake % =

Absorbancetreated group − Absorbanceblank × 100% Absorbancepositive control

In the equation, the treated groups represent the cells treated with GO, rGO, nZVI, or rGO-nZVI NH at 5 µg/mL, the blank represents untreated cell lysate, and positive controls are GO, rGO, nZVI and rGO-nZVI NH in the NaOH-DMSO mixture with no cells. Each sample had three replicates and the mean ± standard deviations were reported.

Confocal microscopy. BEAS-2B cells were treated with GO, nZVI, and rGO-nZVI NH at 25 µg/mL concentration. At 48 h post-treatment, the cells were fixed with 4% paraformaldehyde and the nuclei was counterstained with Hoechst 33342 (Invitrogen, Carlsbad, CA). The cellular uptakes of GO, nZVI, and rGO-nZVI NH in these fixed cells were observed using an LSM 710 confocal laser scanning microscope (Carl Zeiss; Dublin, CA).

Scanning electron microscopy.

Scanning Electron Microscopy (SEM) was used to

investigate the biophysical interaction between BEAS-2B cells and the nanomaterials i.e., GO, rGO, nZVI, and rGO-nZVI NH. For this, BEAS-2B cells were seeded onto cover glasses in 6-well plates at 1.5×105 cells per well and allowed to grow overnight. The cells were treated with GO, rGO, nZVI, and rGO-nZVI NH at concentrations of 15 µg/mL. At 48

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h post-treatment, the cells were washed with PBS twice, and then fixed with 2% glutaraldehyde for 1.5 h at 4 ℃. After fixation, the cells were rinsed with PBS twice, and then sequentially dehydrated with 30%, 50%, 70%, 85%, 95%, and 100% ethanol, 15 min in each step. After dehydration, the cells were incubated with 100% hexamethyldisilazane, dried overnight at room temperature, and imaged by a Hitachi SU70 Field Emission Scanning Electron Microscope (FESEM).

Cell viability study.

BEAS-2B cells were seeded in 12-well plates at 7.5×104 cells per well

and allowed to grow overnight. Then, GO, rGO, nZVI, and rGO-nZVI NH were added to the cells at concentrations of 0 (untreated controls), 5, 10, 15, 25, 35, 50, and 75 µg/mL. At 48 h of post exposure, cells were detached by trypsin and the number of viable cells was counted using trypan blue exclusion method. Each sample had three replicates and the mean ± standard deviations were reported.

Lactate dehydrogenase (LDH) cytotoxicity test.

LDH cytotoxicity of GO, rGO, nZVI, and

rGO-nZVI NH was measured using the Pierce™ LDH Cytotoxicity Assay Kit following manufacturer’s protocol. Briefly, BEAS-2B cells were seeded in 96-well plates at 104 cells per well and allowed to grow overnight. The cells were challenged with GO, rGO, nZVI, and rGO-nZVI NH at concentrations of 0 (untreated control), 5, 15, 25, 50, and 75 µg/mL. At 48 h of post-treatment, 50 µL cell culture medium was transferred into a new 96-well plate. 50 µL reaction mixture was added to the medium and incubated at room temperature for 30 min in dark. Then 50 µL stop solution was added to stop the reaction. The light absorbance at 490

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nm and 680 nm (background signal from instrument) was measured via a Tecan microplate reader (San Jose, CA) and used to calculate the LDH cytotoxicity. Each sample had three replicates and the mean ± standard deviations were reported.

Measurement of intracellular reactive oxygen species (ROS).

The oxidant-sensitive dye

H2DCFDA was used for ROS detection following manufacturer’s protocol. BEAS-2B cells were seeded in the 96-well plates at 104 cells per well and allowed to grow overnight. The cells were challenged with GO, rGO, nZVI, and rGO-nZVI NH at concentrations of 0 (untreated control), 5, 15, 25, 50, and 75 µg/mL. At 24 h post-treatment, cells were washed with PBS twice. Then fresh cell culture medium containing 10 µM H2DCFDA was added and incubated with cells for 30 min at 37 oC, protected from light. Then H2DCFDA was removed by washing the cells with PBS twice. The fluorescence intensities of the cells were measured using the micro-plate reader (λex =485 nm and λem = 535 nm). The ROS level was calculated using the following equation:

ROS fold =

!"#! − !"#!$%&'()

*+(!,+& − *+(!,+&$%&'()

In this equation, Itest represents the fluorescence intensity of nanomaterial-exposed cells treated with H2DCFDA, Itest-blank is the fluorescence intensity of nanomaterial-exposed cells with no H2DCFDA treatment, Icontrol is the fluorescence intensity of control cells treated with H2DCFDA, and Icontrol-blank is the fluorescence intensity of control cells alone without H2DCFDA treatment.

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Cell cycle analyses.

BEAS-2B cells were seeded in the 6-well plates at 1.5×105 cells per

well and allowed to grow overnight. The cells were treated with GO, rGO, nZVI, and rGO-nZVI NH at concentrations of 0 (untreated control), 5, 15, 25, 50, and 75 µg/mL. At 48 h post-treatment, the cells were harvested and fixed in 200 proof ethanol at -20ºC for 1 h. Then the cells were washed with PBS and dispersed in staining buffer (100 mM Tris, pH 7.4, 150 mM NaCl, 1 mM CaCl2, 0.5 mM MgCl2) containing 3µm propidium iodide (PI) and 50 µg/mL RNase A. The fluorescence intensity of cells was measured by BD LSR Fortessa flow cytometer (BD Biosciences, San Jose, CA) using the PI channel. Total 10000 events were recorded.

Statistical analysis.

All results were presented as mean ± standard deviation. The

significance was determined by p-value calculated by t-test. The p values of 0.9) with hydrodynamic diameters varying widely for GO within the range of 150-3000 nm, for rGO within the range of 120-2500 nm, and for nZVI within the range of 2000-7000 nm. It is important to note that although dry nZVIs had smaller particle sizes than GO’s lateral sizes as observed by HRTEM imaging, in DI water nZVIs aggregate highly due to their high van der Waals and magnetic attraction forces and the hydrodynamic diameters ranged in the microns. rGO-nZVI NH in DI water, on the other hand, showed less polydispersity (PDI~0.7) with an initial average hydrodynamic diameter of ~1344±567 nm, however, rGO-nZVI NH didn’t remain colloidally stable in DI water over time and showed a linear increase in hydrodynamic diameter with time. When these nanomaterials (i.e., GO, rGO, nZVI, and rGO-nZVI) were placed in the mixture of dispersion medium and cell culture medium, the hydrodynamic diameters reduced to 361±95, 300±170, 1231±396, and 574±217 nm for GO, rGO, nZVI, and rGO-nZVI, respectively, accompanied by a significantly reduced PDI of ~0.4. The narrower range of hydrodynamic diameters suggests that the dispersion and cell culture media provides better colloidal stability for all the nanomaterials than DI water. However, in the case of nZVI, comparatively wider range of hydrodynamic diameters than GO, rGO, and rGO-nZVI after 1 h suggests aggregation and settling of nZVI during the 1 h period of TR-DLS measurement. More importantly, the results showing smaller

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hydrodynamic diameters of GO, rGO, and rGO-nZVI NH compared to nZVI suggested that graphene support helped reduce the aggregation of nZVI in case of the NH resulting in better colloidal stability. The improved colloidal stability of the nanomaterials in dispersion and culture media compared to DI water can be attributed to adsorption of albumin protein on the surface of the nanomaterials.24-25 Protein corona formation on the surface of the nanomaterials helps in de-agglomerating and resisting further aggregation through steric and

Hydrodynamic Diameter (nm)


electrostatic repulsion.26-27 4000 3500

GO (DI) GO (Dispersion+Culture Medium)

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(a)

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(c)

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Time (minutes)

Figure 2. Aggregation behavior of (a) GO, (b) rGO, (c) nZVI, and (d) rGO-nZVI NH in DI water and dispersion+cell culture medium. The sedimentation profiles of GO, rGO, nZVI, and rGO-nZVI NH in the dispersion media were determined at room temperature and are presented in Figure 3(a). nZVI had the

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highest settling tendency followed by rGO-nZVI NH, GO, and rGO which is in agreement with the similar trend of hydrodynamic diameter values over time from the TR-DLS results mentioned above. Because of the highest density and the largest aggregate size, nZVI settled out of dispersion media at the highest rate among all the four nanomaterials, i.e. almost all nZVI precipitated at 30 min post sonication. However, fractions of rGO-nZVI NH (~10%), GO (~20%), and rGO (~50%) remained suspended even after 48 h.

Cellular uptake.

To investigate the interaction of GO, rGO, nZVI, and rGO-nZVI NH with

biological systems, we first performed the cellular uptake study in BEAS-2B cells. For this study, BEAS-2B cells were treated with these nanomaterials at 5 µg/mL and the amount of nanomarterials uptaken by cells were measured at 1, 2, 4, 8, 12, 24, and 48 h post-exposure. As shown in Figure 3(b), fast cellular uptake of all four nanomatrials was observed during the first 8 h. During that time, nZVI showed the highest cellular uptake rate followed by rGO-nZVI NH, rGO, and GO. In the following 40 h, the cellular uptake of rGO-nZVI NH happened at a steady rate, however, the cellular uptake of GO, rGO, and nZVI slowed down significantly. At 48 h post exposure, ~84.5% rGO-nZVI NH, ~70.3% nZVI, ~50% rGO, and ~40% GO were taken up by the cells. Confocal microscopy images post 48 h confirmed that the nanomaterials were successfully taken up by the cells and accumulated inside the cells as shown with red arrows within Figure S3.

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GO rGO nZVI rGO-nZVI

0.8 0.6 0.4 0.2

(b)

GO rGO nZVI rGO-nZVI

100

Cellular Uptake (%)

(a)

1.0

Sediment Function (Ft)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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80 60 40 20

0.0 0 0

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Figure 3. (a) Sedimentation profile of GO, rGO, nZVI, and rGO-nZVI in the dispersion media. (b) Cellular uptake of GO, rGO, nZVI, and rGO-nZVI NH in BEAS-2B cells. Concentration of each material was 5 µg/mL. The differences in the cellular uptake and its rates for GO, rGO, nZVI, and rGO-nZVI can be explained by the aggregation and sedimentation behavior of the nanomaterials in the biological media as discussed above. The high cellular uptake rate of nZVI in the initial hours can be attributed to the fast sedimentation of nZVI which led to the high availability of nZVI to the cells attached on the surface.28 rGO showed the slowest sedimentation and smallest aggregate sizes which rendered the second lowest cellular uptake rate. Although GO showed faster sedimentation than rGO, the aggregate sizes of GO were larger than those of rGO and this difference resulted in the lowest cellular uptake for GO. The sedimentation rate and aggregate size of rGO-nZVI NH were in between those of GO and nZVI; this enabled continuous availability to cells, and thus showed the highest cellular uptake post 48 h. Compared to nZVI aggregates (1231±396 nm), rGO-nZVI NH aggregates had significantly smaller size (574±217 nm), which may facilitate more cellular uptake of rGO-nZVI NH, therefore higher amount of rGO-nZVI NH (~84.5%) than nZVI (~70.3%) was observed

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inside the cells at 48 h post treatment. Besides the dominance of sedimentation on the cellular uptake in the initial hours, several other mechanisms (e.g., particle size,29 shape,30 surface charge31) may have governed the cellular uptake process. The higher uptake of rGO-nZVI NH compared to the parent materials suggests synergistic and modified effect in cellular interaction. Many reasons including increased hydrophobicity of graphene due to reduction during hybridization,29 folded and wrinkled shapes of graphene in the NH32 can contribute in higher uptake of rGO-nZVI NH.

Biophysical interaction between the nanomaterials and BEAS-2B cells.

SEM was used to

further elucidate the biophysical interaction between the nanomaterials and the cells. BEAS-2B cells were treated with 15 µg/mL GO, rGO, nZVI, and rGO-nZVI NH for 48 h. SEM images presented in Figures 4, S4, and S5 helped to differentiate the effect of hybridization on the cell-nanomaterial interactions. The changes of cell morphology were similar for cells treated with rGO-nZVI NH and nZVI, however, those treated with GO (and rGO) presented with different morphology. GO and rGO showed plate-shaped structures and seemed to have disrupted the cells physically. Some GO and rGO nanosheets aligned and attached themselves in parallel to the cell membrane, and some GO and rGO slid in the cell membrane (indicated with arrows in the Figures). Previous literature reports also support these observations: GO and rGO were able to either align parallel to or pierced through the membranes of bacteria,33-35 human hepatocellular carcinoma HepG2 cells,36 Caco-2 human intestinal cells,37 human and murine macrophages,38 and mouse mesenchymal progenitor C2C12 cells.39 Such penetrations of cell membranes by the GO sheets are found to be due to

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the interaction with the blade-like edges of GO sheets.34,

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GO (or rGO) nanosheets

sometime can wrap the cells causing physical damage,42-43 however, we didn’t observe such cases in our study. Among other nanomaterials, rGO-nZVI NH and nZVI were found to be agglomerated and integrated within the cells (indicated with arrows in Figures 4 and S4), and were possibly up-taken through endocytosis. While our study first revealed the interaction between rGO-nZVI NH and the cell membranes, our observations regarding nZVI-cell interactions agreed well with results reported previously for nZVI. Aggregates of nZVI particles were found inside bacterial cell membrane that disrupted the membrane integrity and reduced bacteria viability.44-45 Because the interaction between rGO-nZVI NH and BEAS-2B cell membrane shared more similarity with nZVI than with GO and rGO, we concluded that the biophysical interaction between rGO-nZVI NH and cells was dominated by nZVI.

Figure 4. Biophysical interaction between BEAS-2B cells and GO, nZVI, and rGO-nZVI NH as seen by SEM. BEAS-2B cells were treated with 15 µg/mL GO, nZVI, and rGO-nZVI NH for 48h.

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Cytotoxic effects of the nanomaterials on the BEAS-2B cells.

We evaluated the in vitro

cytotoxicity of GO, rGO, nZVI, and rGO-nZVI NH at various concentrations ranging from 0 to 75 µg/mL upon exposure to BEAS-2B by cell viability enumeration. At 48 h post treatment, the numbers of viable cells were counted using trypan blue exclusion method. As shown in Figure 5(a), the exposure to all four nanomaterials caused toxicity and reduced viable cell numbers, however, in a material type-dependent and dose-dependent manner. The higher the dosage, the lower the viable cell numbers and hence the higher is the toxicity. Similar dose-dependent toxicity of GO, rGO, and nZVI was reported previously,12,

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however, this is the first report of dose-dependent cellular toxicity for rGO-nZVI NHs. In terms of material type, GO and rGO showed the highest toxicity followed by rGO-nZVI NH, and nZVI showed the lowest cytotoxic effect. Thus, the hybridization of nZVI with GO (or rGO) increased the toxicity of nZVI. The half maximal inhibitory concentration or IC50 value of rGO-nZVI (27 µg/mL) was closer to that of GO and rGO (17 µg/mL) than nZVI (67 µg/mL), suggesting that the toxicity of rGO-nZVI NH was mainly induced by carbonaceous component i.e., GO or rGO.

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(a)

(b)

(c)

(d)

Figure 5. (a) Viable cell number of BEAS-2B cells at 48h post-exposure of GO, rGO, nZVI, and rGO-nZVI NH at concentrations ranging from 0 to 75 ug/mL. (b) LDH cytotoxicity of GO, rGO, nZVI, and rGO-nZVI NH in BEAS-2B cells at 48 h post-treatment. (c) Intracellular ROS levels of BEAS-2B cells at 24 h post-exposure to GO, rGO, nZVI, and rGO-nZVI NH. (d) Percentages of BEAS-2B cells in G1 phase at 48 h post exposure of GO, rGO, nZVI, and rGO-nZVI NH. ‘*’ denotes the significance level of the statistical analyses as p

In Vitro Pulmonary Toxicity of Reduced Graphene Oxide-Nano Zero

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