Live Imaging of Label-Free Graphene Oxide ... - ACS Publications

Dec 29, 2017 - The interest in graphene and its translation into commercial products has been expanding at a high pace. Based on previously described ...
0 downloads 0 Views 3MB Size
Subscriber access provided by RMIT University Library

Article

Live Imaging of Label-Free Graphene Oxide Reveals Critical Factors Causing Oxidative Stress-Mediated Cellular Responses Sandra Vranic, Artur Filipe Rodrigues, Maurizio Buggio, Leon Newman, Michael R. H. White, David G. Spiller, Cyrill Bussy, and Kostas Kostarelos ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b07734 • Publication Date (Web): 29 Dec 2017 Downloaded from http://pubs.acs.org on December 30, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Nano is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 52 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

ACS Nano

Live Imaging of Label-Free Graphene Oxide Reveals Critical Factors Causing Oxidative Stress-Mediated Cellular Responses Sandra Vranic*, 1, 2, Artur Filipe Rodrigues*, 1, 2, Maurizio Buggio1, 2, Leon Newman1, 2, Michael R. H. White3, David G. Spiller4, Cyrill Bussy1, 2, # and Kostas Kostarelos1, 2, # 1 Nanomedicine Laboratory, School of Health Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, AV Hill Building, Manchester M13 9PT, UK. 2

National Graphene Institute, The University of Manchester, Booth Street East, Manchester M13 9PL, UK.

3

School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Michael Smith Building, Manchester M13 9PT, UK. 4

FBMH Platform Sciences, Enabling Technologies & Infrastructure, FBMH Research & Innovation, Faculty of Biology, Medicine and Health, The University of Manchester, Michael Smith Building, Manchester M13 9PT, UK.

________________________________ #

Corresponding to: [email protected]; [email protected]

* These authors contributed equally to this study.

ORCiD numbers: Sandra Vranic # 0000-0002-6653-7156 Artur Rodrigues # 0000-0002-4078-3455 Maurizio Buggio # 0000-0001-9884-6255 Leon Newman # 0000-0003-2099-2660 David G. Spiller # 0000-0003-2502-6787 Michael R. H. White # 0000-0002-3617-3232 Cyrill Bussy # 0000-0001-8870-443X Kostas Kostarelos # 0000-0002-2224-6672

1 ACS Paragon Plus Environment

ACS Nano 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

Page 2 of 52

Abstract The interest in graphene and its translation into commercial products have been expanding at high pace. In regard to previously described pulmonary safety concerns for carbon nanomaterials, there is a great need to define parameters guiding interactions between graphenebased materials and pulmonary system. The aim of present study was to determine the importance of two critical parameters: lateral dimensions of the material and coating with proteins in relation to each other and their pulmonary impact. Endotoxin-free materials with distinct lateral dimensions – s-GO (50 – 200 nm) and l-GO (5 – 15 µm) were produced and thoroughly characterized. Exploiting intrinsic fluorescence of GO and using confocal live-cell imaging, we visualized the behavior of the cells in response to the material in real time. Although BEAS-2B cells internalized GO efficiently, lGO was linked to higher plasma membrane interactions correlated with elevated ROS levels, proinflammatory response and greater cytotoxicity, in agreement with the oxidative stress paradigm. For both GO types, the presence of serum alleviated lipid peroxidation of plasma membrane and decreased intracellular ROS levels. However, protein coating was not enough to entirely mitigate toxicity and inflammatory response induced by l-GO. In vitro results were validated in vivo, as l-GO was more prone to induce pulmonary granulomatous response in mice compared to s-GO. In conclusion, lateral dimension of GO played more important role than serum protein coating in determining biological responses to the material. It was also demonstrated that time-lapse imaging of live cells interacting with label-free GO sheets can be used as a tool to assess GO induced cytotoxicity. Keywords: 2D materials, graphene, confocal live cell imaging, inflammation, lungs, nanotoxicology

2 ACS Paragon Plus Environment

Page 3 of 52 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

ACS Nano

The interest in graphene and its translation into commercial products have been expanding during the last few years. Graphene-based materials (GBMs) are a large family of different materials1, 2 varying tremendously in their lateral dimensions, number of carbon layers (i.e. thickness) and surface properties. Today, GBMs are already contained in various commercial products (sportswear, tennis rackets, bicycle frames, tires, or innovative paints) while conductive inks, paints or filaments for 3D printing are expected to become components of flexible displays and wearable electronics in the near future.3 Longer-term applications are also anticipated in the healthcare sector, either as drug delivery systems, biosensors for health monitoring and e-health, or in the form of biomedical implants.2 However, in order to be fully adopted by both industry and society, new enabling technologies need to demonstrate not only their benefits but also their longterm safety and sustainability.2, 4 In consideration of the previously described pulmonary health and safety concerns for carbon nanomaterials (e.g. carbon nanotubes) 5, 6 and the fact that the lungs will be one of the primarily exposed organs following inhalation of nanomaterials (in particular airborne GBMs), there is a great need to understand the critical parameters impacting interactions between GBMs and the cells of the pulmonary system. For the vast majority of GBMs, interactions with mammalian cells and their overall safety profile remain largely unknown. By far the most studied type of GBMs in biological research has been graphene oxide (GO), the heavily oxidized form of graphene in the form of sheets. This popularity can be explained by its ease of production and its hydrophilic surface character that allows its colloidal dispersion in aqueous and physiological milieu. Hazard assessment studies have indicated that structure-function relationships should be based on specific physicochemical properties of the studied nanomaterial. Both intrinsic (e.g. physicochemical properties) and acquired (e.g. aggregation state in local milieu or secondary coating with components from the cell culture medium, blood or different organs) features need to be taken into consideration before obtaining clear understanding of the parameters that could lead to adverse effects.7 In addition, the oxidative stress paradigm has been shown to successfully explain and predict the outcomes of the cellular response to many nanomaterials.8-10 According to this paradigm, cellular response to nanomaterials is governed by induction of oxidative stress that can be either counter-balanced by the activity of anti-oxidative enzymes (tier 1 response), lead to subsequent activation of pro-inflammatory pathways (tier 2 response) or result in cell death in the case of excessive levels of reactive oxygen species (ROS) production (tier 3 response). Previous in vitro studies have compared graphene oxide sheets (GOs) of large (2 µm) and small (350 nm) lateral dimensions to conclude that larger materials were more inflammogenic than smaller ones, with an almost flat inflammatory response for the small GO even at the highest concentration (6 µg/mL) tested.11 A different study by Ma et al. reported that large GO (750-1300 nm) induced more cytokine production than two smaller GO types (50-350 nm or 350-750 nm), but

3 ACS Paragon Plus Environment

ACS Nano 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

Page 4 of 52

concluded that all GOs were inflammogenic.12 GO sheets were also found to be pro-fibrotic in cells and animals, with larger GO (1676 nm) sheets being more potent than their smaller counterparts (179 nm).5 Currently, only a limited number of studies have reported that small GO may cause more damage than larger GO (above 1 µm). Orecchioni et al. found smaller GO ( 3 µm) is not well documented, as most studies have only considered GO nano-sheets (