PEGylated gold nanorods as optical trackers for biomedical applications: an in vivo and in vitro comparative study Gaser N Abdelrasoul 1,4 , Raffaella Magrassi 1,3,4 , Silvia Dante 1 , Marta d’Amora 1 , Marco Scotto d’Abbusco 1 , Teresa Pellegrino 1 and Alberto Diaspro 1,2 1 Istituto Italiano di Tecnologia, via Morego 30, 16163 Genova, Italy 2 Department of Physics, University of Genova, via Dodecaneso 33, 16146 Genova, Italy 3 Biophysics Institute, National Research Council (CNR), via De Marini 6, 16149 Genova, Italy E-mail: alberto.diaspro@iit.it Received 11 September 2015, revised 13 April 2016 Accepted for publication 26 April 2016 Published 13 May 2016 Abstract Gold nanorods (AuNRs) are eligible for a variety of biological applications including cell imaging, sensing, and photothermal therapy thanks to their optical properties. The aim of this work is to show how AuNRs could be employed as non-photobleachable optical contrast agents for biomedical applications. In order to demonstrate the feasibility of their use as optical trackers, we employed two-photon emission confocal microscopy on cells incubated with PEGylated AuNRs. Remarkably, AuNRs were localized mostly in the perinuclear zone and microscopy characterization showed the presence of a considerable number of rods inside cell nuclei. Furthermore, we estimated the toxicity and the efficiency of cellular uptake of the PEGylated AuNRs as a function of administered dose on HeLa/3T3 cell lines and on zebrafish during development, employed as an in vivo model. Eventually, we observed good agreement between in vivo and in vitro experiments. The employed AuNRs were prepared through a photochemical protocol here improved by tuning the amount of the cationic surfactant cetyltrimethylammonium bromide for the achievement of AuNRs at two different aspect ratios. Furthermore we also investigated if the AuNR aspect ratio influenced the toxicity and the efficiency of cellular uptake of the PEGylated AuNRs in HeLa/3T3 cell lines and in zebrafish embryos. S Online supplementary data available from stacks.iop.org/NANO/27/255101/mmedia Keywords: gold nanorods, two-photon excitation, optical tracking, photochemical synthesis, cell viability test, in vivo zebrafish test, cell proliferation test (Some figures may appear in colour only in the online journal) 1. Introduction The need for biocompatible and non-photobleachable optical contrast agents is a strong motivation to investigate nanos- tructured materials possessing distinguishable optical prop- erties [1]. In recent years gold nanoparticles (AuNPs) have drawn the attention of researchers in nanobiotechnology due to their distinctive optical properties originating from the interaction of incident light with the electromagnetic field arising from the movement of their conduction band electrons [2]. In the literature, various methodologies have been exploited to produce AuNPs with tunable sizes and diverse morphology, including spheres [3], rods [4], hexapods [5], and stars [6]. Furthermore, gold nanorods (AuNRs) have emerged as good candidates for several biological applica- tions such as cell imaging, drug delivery, and photothermal therapy [7]. AuNRs possess the particular optical property that their dispersion is depicted by two surface plasmonic Nanotechnology Nanotechnology 27 (2016) 255101 (15pp) doi:10.1088/0957-4484/27/25/255101 4 These authors contributed equally to this work. 0957-4484/16/255101+15$33.00 © 2016 IOP Publishing Ltd Printed in the UK 1