Aqueous cationic, anionic and non-ionic multi-walled carbon nanotubes, functionalised with minimal framework damage, for biomedical application Shu Chen a,1 , Sheng Hu b,1 , Elizabeth F. Smith c , Pakatip Ruenraroengsak a , Andrew J. Thorley c , Robert Menzel b , Angela E. Goode a , Mary P. Ryan a , Teresa D. Tetley c , Alexandra E. Porter a , Milo S.P. Shaffer b, * a Department of Materials and London Centre for Nanotechnology, Imperial College London, Exhibition Road, London SW7 2AZ, UK b Department of Chemistry and London Centre for Nanotechnology, Imperial College London, Exhibition Road, London SW7 2AZ, UK c National Heart and Lung Institute, Imperial College London, Dovehouse Street, London SW3 6LY, UK article info Article history: Received 6 January 2014 Accepted 4 February 2014 Available online 14 March 2014 Keywords: Multiwalled carbon nanotubes Thermal chemical functionalisation Integrity structure Surface charge Cytotoxicity Cell uptake abstract The use of a thermochemical grafting approach provides a versatile means to functionalise as- synthesised, bulk multi-walled carbon nanotubes (MWNTs) without altering their inherent structure. The associated retention of properties is desirable for a wide range of commercial applications, including for drug delivery and medical purposes; it is also pertinent to studies of intrinsic toxicology. A systematic series of water-compatible MWNTs, with diameter around 12 nm have been prepared, to provide structurally-equivalent samples predominantly stabilised by anionic, cationic, or non-ionic groups. The surface charge of MWNTs was controlled by varying the grafting reagents and subsequent post- functionalisation modifications. The degree of grafting was established by thermal analysis (TGA). High resolution transmission electron microscope (HRTEM) and Raman measurements confirmed that the structural framework of the MWNTs was unaffected by the thermochemical treatment, in contrast to a conventional acid-oxidised control which was severely damaged. The effectiveness of the surface modification was demonstrated by significantly improved solubility and stability in both water and cell culture medium, and further quantified by zeta-potential analysis. The grafted MWNTs exhibited rela- tively low bioreactivity on transformed human alveolar epithelial type 1-like cells (TT1) following 24 h exposure as demonstrated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium (MTS) and lactate dehydrogenase release (LDH) assays. The exposure of TT1 cells to MWNTs suppressed the release of the inflammatory mediators, interleukin 6 (IL-6) and interleukin 8 (IL-8). TEM cell uptake studies indicated efficient cellular entry of MWNTs into TT1 cells, via a range of mechanisms. Cationic MWNTs showed a more substantial interaction with TT1 cell mem- branes than anionic MWNTs, demonstrating a surface charge effect on cell uptake. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction The biological interactions of carbon nanotubes (CNTs) are important for fundamental scientific studies [1,2], biomedical ap- plications such as photothermal therapy [3,4], drug delivery [5,6] and bioimaging [7,8], and in order to understand their potential toxicities [5,9,10]. The poor solubility and lack of control over the physicochemical properties of CNTs have created significant ob- stacles to understanding their interactions with cells. Delivery of individualised CNTs either in vitro in cell medium or in vivo is rare, particularly for toxicology studies, in which agglomerated or even non-aqueous dispersions are commonly used. Surfactants, such as Triton X-100 [5,6,11] and sodium dodecyl sulphate (SDS) [7,8,12], can be used to stabilise aqueous dispersions but use is limited by surfactant toxicity which can cause confounding effects [9,10,13] and do not relate to many real life situations. In addition, surfac- tant dispersions are meta-stable, with a surface chemistry that evolves over time, and are generally unsuitable for in vivo use [14]. Where modified, water-stabilised CNTs have been used, the * Corresponding author. E-mail address: m.shaffer@imperial.ac.uk (M.S.P. Shaffer). 1 These authors contributed equally to this work. Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials http://dx.doi.org/10.1016/j.biomaterials.2014.02.002 0142-9612/Ó 2014 Elsevier Ltd. All rights reserved. Biomaterials 35 (2014) 4729e4738