SANDIN ET AL. VOL. 6 ’ NO. 2 ’ 1513–1521 ’ 2012 www.acsnano.org 1513 January 25, 2012 C 2012 American Chemical Society High-Speed Imaging of Rab Family Small GTPases Reveals Rare Events in Nanoparticle Trafficking in Living Cells Peter Sandin, †, * Laurence W. Fitzpatrick, † Jeremy C. Simpson, ‡ and Kenneth A. Dawson †, * † Centre for BioNano Interactions, School of Chemistry and Chemical Biology & UCD Conway Institute of Biomolecular and Biomedical Research and ‡ School of Biology and Environmental Science & UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland D ue to their potential as tools to im- prove diagnosis and treatment of diseases, engineered nanoparticles (NPs) have received much attention in recent years. However, despite significant efforts, considerable uncertainties remain about the mechanisms by which NPs are taken up and trafficked in cells (for recent reviews summarizing published data as well as discussing different approaches currently employed, see refs 1À6). Studies on the endocytic mechanisms potentially used have shown NP uptake to be dependent on NP size, 7À9 shape, 7,10 surface, 8,10À12 and cell type. 11À14 On the other hand, studies on intracellular trafficking have shown that most NPs, irrespective of shape, charge, and size, accumulate in the lysosomes, although there are examples indicating that NP escape from this degradative pathway is possible. 9,11,15,16 To date, most studies have focused on identifying the point of entry and final destination; however, for NPs to meet their promise in the field of nanomedi- cine, delivery to desired organs and cell types will not be adequate, but rather the ability to target specific subcellular compartments will be needed. To realize this, a deeper knowledge and understanding of the entire intracellular itinerary will be essential, and in the nanoma- terial and NP field, this is a considerable chal- lenge. Unlike biomolecules, NPs can be constructed from a wide variety of materials, therefore making their surfaces notoriously heterogeneous in structure, in turn resulting in a diversity of biological molecules (their biomolecular corona) that can be bound to them. 17À20 It is, in principle, possible that the original corona, combined with new molecules picked up inside the cell, could, even if rarely, provide the NPs with signals to allow them to access di fferent subcellular destinations. Stud- ies addressing this critical concept have com- monly used fluorescence microscopy on fixed cells to analyze cellular localization and distri- bution of NPs. However, clearly such ap- proaches will only ever provide a snapshot representing their bulk movement inside the cell. This will mean that minor, but potentially important, subpopulations of NPs will be over- looked. In addition, such approach is also known to potentially cause fixation artifacts, particularly in regard to spatial organization and distribution. 21 * Address correspondence to psandin@chalmers.se, kenneth.a.dawson@cbni.ucd.ie. Received for review November 16, 2011 and accepted January 25, 2012. Published online 10.1021/nn204448x ABSTRACT Despite the increased application of nanomaterials in diagnostics and therapeutics, methods to study the interactions of nanoparticles with subcellular structures in living cells remain relatively undeveloped. Here we describe a robust and quantitative method that allows for the precise tracking of all cell-associated nanoparticles as they pass through endocytic compart- ments in a living cell. Using rapid multicolor 3D live cell confocal fluorescence microscopy, combined with transient overexpression of small GTPases marking various endocytic membranes, our studies reveal the kinetics of nanoparticle trafficking through early endosomes to late endosomes and lysosomes. We show that, following internalization, 40 nm polystyrene nanoparticles first pass through an early endosome intermediate decorated with Rab5, but that these nanoparticles rapidly transfer to late endosomes and ultimately lysosomes labeled with Rab9 and Rab7, respectively. Larger nanoparticles of 100 nm diameter also reach acidic Rab9- and Rab7-positive compartments although at a slower rate compared to the smaller 40 nm nanoparticles. Our work also reveals that relatively few nanoparticles are able to access endocytic recycling pathways, as judged by lack of significant colocalization with Rab11. Finally, we demonstrate that this quantitative approach is sufficiently sensitive to be able to detect rare events in nanoparticle trafficking, specifically the presence of nanoparticles in Rab1A-labeled structures, thereby revealing the wide range of intracellular interactions between nanoparticles and the intracellular environment. KEYWORDS: nanoparticles . membrane traffic . Rab GTPases . intracellular trafficking . colocalization . live cell imaging . polystyrene particles ARTICLE