Intracellular trafficking of superparamagnetic iron oxide nanoparticles conjugated with TAT peptide: 3-dimensional electron tomography analysis Baiju G. Nair, Takahiro Fukuda, Toru Mizuki, Tatsuro Hanajiri, Toru Maekawa ⇑ Bio-Nano Electronics Research Centre, Toyo University, Saitama 350-8585, Japan article info Article history: Received 28 March 2012 Available online 21 April 2012 Keywords: Nanoparticles Internalisation Cell penetrating peptides Endocytic pathways Transmission electron microscopy 3-D tomography abstract Internalisation of nanoparticles conjugated with cell penetrating peptides is a promising approach to var- ious drug delivery applications. Cell penetrating peptides such as transactivating transcriptional activator (TAT) peptides derived from HIV-1 proteins are effective intracellular delivery vectors for a wide range of nanoparticles and pharmaceutical agents thanks to their amicable ability to enter cells and minimum cytotoxicity. Although different mechanisms of intracellular uptake and localisation have been proposed for TAT conjugated nanoparticles, it is necessary to visualise the particles on a 3-D plane in order to inves- tigate the actual intracellular uptake and localisation. Here, we study the intracellular localisation and trafficking of TAT peptide conjugated superparamagnetic ion oxide nanoparticles (TAT-SPIONs) using 3-D electron tomography. 3-D tomograms clearly show the location of TAT-SPIONs in a cell and their slow release from the endocytic vesicles into the cytoplasm. The present methodology may well be uti- lised for further investigations of the behaviours of nanoparticles in cells and eventually for the develop- ment of nano drug delivery systems. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction Nanoparticles internalised in cells can be utilised in various bio- medical areas such as hyperthermia, drug release, imaging and gene silencing [1–3]. To make the above operations successful, nanoparticles need to enter the cells and stay inside for a period of time, which in particular projects an important aspect of nano- drug delivery systems [4]. It is well known that nanoparticles can enter a cell via different methods; i.e., non specific uptake by endo- cytosis, direct injection of nanomaterials, electroporation and spe- cifically targeted uptake of nanomaterials functionalised with various targeting moieties. Among the above methods, nanoparti- cles conjugated with specific ligands are highly promising for selective nanodrug delivery systems [5]. Cell penetrating peptide (CPP) is supposed to be one of the best ligands to improve active internalisation of nanoparticles into a target cell [6]. CPPs in gen- eral translocate efficiently across cell membranes, but the exact mechanism of the cell penetrating activity is still under investiga- tion [7]. CPPs such as transactivating transcriptional activator (TAT) peptide derived from HIV can transport a large number of nanoparticles into a mammalian cell cytoplasm avoiding the nor- mal endocytic pathways through the penetration of the cell mem- brane [8]. Complete lack of specificity of TAT peptides used to be a solemn matter of concern for targeting a cancer cell, but a selective internalisation of nanoparticles into a cancer cell was successfully performed by combining TAT peptides with cell targeting peptides (CTPs) [9]. This kind of combined peptides can be utilised for the internalisation of an enormous amount of nanoparticles into cells and are also useful for various intracellular manipulation studies [10]. Internalisation of a massive number of nanoparticles into a target cell and their escape from the normal endocytic pathways are attractive features particularly for nano drug delivery systems [11]. Most of the studies on the internalisation of nanoparticles are based on the confocal and flow cytometry analyses. However, a complete visualisation of intracellular trafficking of nanoparticles using the above optical methods is quite difficult due to the high electrostatic interaction between nanoparticles and the surface of cells, which often leads to wrong interpretations of the data [12]. Transmission electron microscopic (TEM) studies support the iden- tification and characterisation of nanoparticles internalised in a cell [13–16]. In the case of 2-D TEM images, however, it is extre- mely difficult to differentiate whether the nanoparticles are pres- ent in the resin or lying on the surface of cell sections without being internalised, or whether the nanoparticles are distributed in the 3-D volume of the section or not. 3-D electron tomography (ET) is a well-established technique in biological sciences. In 3-D ET, the specimen is rotated around an axis at a range of tilt angles perpendicular to the electron beam and multiple 2-D projection images of a 3-D sample are recorded. Finally, high resolution 3-D images are constructed from a set of 2-D images [17]. The 3-D structures of organelles and macromolecular assemblies [18] and the molecular organisation of the cytoplasm in thin sections of 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbrc.2012.04.080 ⇑ Corresponding author. E-mail address: maekawa@toyo.jp (T. Maekawa). Biochemical and Biophysical Research Communications 421 (2012) 763–767 Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc