CASTLE ET AL . VOL. 5 ’ NO. 4 ’ 2458–2466 ’ 2011 www.acsnano.org 2458 March 02, 2011 C 2011 American Chemical Society Hydroxyl-Functionalized and N-Doped Multiwalled Carbon Nanotubes Decorated with Silver Nanoparticles Preserve Cellular Function Alicia B. Castle, † Eduardo Gracia-Espino, ‡ C esar Nieto-Delgado, ‡ Humberto Terrones, ) Mauricio Terrones, §,^, * and Saber Hussain †, * † AFRL/711HPW/RHPB, Wright-Patterson Air Force Base, Ohio 45433-5707, United States, ‡ Advanced Materials Department, IPICYT, Camino a la Presa San Jos  e 2055, San Luis Potosí 78216, M  exico, § Research Center for Exotic Nano Carbons (JST), Shinshu University, 4-17-1 Wakasato, Nagano City 380-8553, Japan, ) Institute of Condensed Matter and Nanosciences (IMCN), Universit  e Catholique de Louvain, Place Croix du Sud 1, B-1348 Louvain-la-Neuve, Belgium, and ^ Department of Physics, Department of Materials Science and Engineering & Materials Research Institute, The Pennsylvania State University, University Park, Pennsylvania 16802-6300, United States B iological functionalization of nanoma- terials has attracted great attention in recent years because of the nanoma- terials application in imaging and signaling pathway detection systems 1 and as a plat- form for the development of biosensors and drug deliverers. Multiwalled carbon nano- tubes (MWNTs) have the unique ability to be functionalized 2 and well dispersed in aqu- eous media with various nanoparticles (e.g., Ag) and chemical groups, and therefore be used as delivery vehicles. However, because of their toxicity there is a great need to develop modification options to increase their biocompatibility. If the nanomaterials are biocompatible, they can be used as specific capture agents, which can be of advantage in biological systems. Ag nanoparticles (Ag-NPs) possess a high extinction coefficient, high surface plasmon resonance and antimicrobial properties and may be less toxic than the bulk form. 3,4 Currently, isolated and well- dispersed Ag-NPs have a variety of uses in everyday life such as Ag-NP infused storage containers, 5 Ag-NPs used to coat medical devices to reduce hospital related infections, 6 and applications in bandages, 7 footwear 8 and countless household items which claim to be antimicrobial. Owing to nanosilver's high surface plasmon resonance, it could be applied to many color-based biosensor ap- plications and different lab-on-a-chip sen- sors. While all of these properties appear to make nano-Ag the new “miracle” of the nanotechnology world, other issues have been developing. Ag-NPs have been shown to be toxic in a size-dependent manner according to Carlson et al. 9 The size dependent toxicity has been shown to be due to reactive oxygen species (ROS) production. A decrease in the mem- brane integrity allows the leakage of ROS outside the cell and leads to apoptosis. 9 Another mechanism of Ag-NP toxicity is believed to be due to the strong affinity of Ag to thiol groups. 10 The thiol groups are the functional groups of the amino acid * Address correspondence to mtterones@shinshu-u.ac.jp, mut11@psu.edu, Saber.hussain@wpafb.af.mil. Received for review May 13, 2010 and accepted March 2, 2011. Published online 10.1021/nn200178c ABSTRACT The present study aims to investigate biocompatibility of silver nanoparticles (Ag- NPs) anchored to different types of multiwalled carbon nanotubes (MWNTs). The MWNTs were decorated with Ag-NPs via a novel chemical route without using any sulfur containing reagent. Three different MWNTs were used as substrate materials for anchoring Ag-NPs: MWNTs-Ag (pure carbon), COx-MWNTs-Ag (carboxyl functionalized), and CNx-MWNTs-Ag (nitrogen-doped). The Ag-NPs, synthesized without thiol capping groups, and which were strongly anchored to the nanotubes surfaces, exhibit an average size of 7 ( 1, 10 ( 1, and 12 ( 1 nm in MWNTs, COx-MWNTs, and CNx- MWNTs, respectively. To determine biocompatibility of these three types of novel hybrid Ag- nanotube materials, cellular function and immune response were evaluated in the human keratinocyte cell line (HaCaT). Cellular assays revealed marginal toxicity after 24 h, and full cellular recovery was observed at 48 h based on an MTS assay for cellular viability. Therefore, Ag-nanotube systems appear to be very different from isolated dispersed Ag-NPs, and due to the strong interactions between the Ag-NPs and the doped nanotube surfaces, they make the Ag particles less toxic because they are not released easily to the cells. Pure carbon MWNTs appear to start releasing Ag-NPs at periods longer than 1 week by an observed decrease in cell proliferation. However, the use of N- and COx-doped MWNTs do not appear to release Ag-NPs to the cells due to the strong binding to the tube surfaces caused by the doped sites. We envisage the use of COx-MWNTs, and CNx-MWNTs anchored with Ag-NP as efficient drug delivery carriers and biosensors. KEYWORDS: Ag nanoparticles . anchoring . nanotubes . biocompatibility . doping . HaCaT . toxicity . drug delivery ARTICLE