Imaging and Tracking of Tat Peptide-Conjugated Quantum Dots in Living Cells: New Insights into Nanoparticle Uptake, Intracellular Transport, and Vesicle Shedding Gang Ruan, ² Amit Agrawal, ² Adam I. Marcus, ‡ and Shuming Nie* ,²,§ Contribution from the Department of Biomedical Engineering, Emory UniVersity and Georgia Institute of Technology, 101 Woodruff Circle, Suite 2001, Atlanta, Georgia 30322, Winship Cancer Institute, Emory UniVersity School of Medicine, Atlanta, Georgia, 30322, and Department of Chemistry, Emory UniVersity, Atlanta, Georgia, 30322 Received July 25, 2007; E-mail: snie@emory.edu Abstract: We report the use of Tat peptide-conjugated quantum dots (Tat-QDs) to examine the complex behavior of nanoparticle probes in live cells, a topic that is of considerable current interest in developing advanced nanoparticle agents for molecular and cellular imaging. Dynamic confocal imaging studies indicate that the peptide-conjugated QDs are internalized by macropinocytosis, a fluid-phase endocytosis process triggered by Tat-QD binding to negatively charged cell membranes. The internalized Tat-QDs are tethered to the inner vesicle surfaces and are trapped in cytoplasmic organelles. The QD loaded vesicles are found to be actively transported by molecular machines (such as dyneins) along microtubule tracks. The destination of this active transport is an asymmetric perinuclear region (outside the cell nucleus) known as the microtubule organizing center (MTOC). We also find that Tat-QDs strongly bind to cellular membrane structures such as filopodia and that large QD-containing vesicles are released from the tips of filopodia by vesicle shedding. These results provide new insights into the mechanisms of Tat peptide-mediated delivery as well as toward the design of functionalized nanoparticles for molecular imaging and targeted therapy. Introduction Bioconjugated semiconductor quantum dots (QDs) are a new class of fluorescent probes under intense research and develop- ment for broad applications in molecular, cellular, and in vivo imaging. 1-6 The basic rationale is that these nanometer-sized particles have unique functional and structural properties, such as size and composition tunable fluorescence emission, large absorption cross sections, and exceptional brightness and photostability compared with organic dyes and fluorescent proteins. Recent research has achieved considerable success in using QDs for labeling fixed cells and tissue specimens and for imaging cell membrane proteins. 7-11 However, only limited progress has been made in developing QD probes for molecular imaging inside living cells. 12-16 A major problem is the lack of efficient methods for delivering monodispersed (that is, single) QDs into the cytoplasm of living cells. A common observation is that QDs tend to aggregate inside living cells and are often trapped in organelles such as vesicles, endosomes, and lysos- omes. As a result, little is known about the interactions of QDs with intracellular proteins and their transport behavior inside living cells. We have used Tat peptide-conjugated QDs (Tat-QDs) as a model system to examine the cellular uptake and intracellular transport of nanoparticles in live cells. Previous work has used cell-penetrating peptides such as polyarginine and Tat to deliver QDs into living cells, 17,18 but the delivery mechanism and the ² Department of Biomedical Engineering, Emory University and Georgia Institute of Technology. ‡ Winship Cancer Institute. § Department of Chemistry, Emory University. (1) Chan, W. C. W.; Maxwell, D. J.; Gao, X. H.; Bailey, R. E.; Han, M. Y.; Nie, S. M. Curr. Opin. Biotechnol. 2002, 13, 40-46. (2) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. 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