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.
"These dynamic events were not clearly synchronized in time and could be observed over a wide duration of time (t = 3–12 min) following pulsed QD-BDNF treatment (1 min). Unlike the linearly-directed trafficking found in neuronal processes or the nuclear-directed trafficking of other intracellular complexes , , QD-BDNF complexes did not show a net directional gain toward any particular subcellular target destination. Instead BDNF complexes could be found meandering over surprisingly long durations (several minutes) and over long distances (up to tens of microns) throughout the neuronal soma. "
[Show abstract][Hide abstract] ABSTRACT: Accumulating evidence underscores the importance of ligand-receptor dynamics in shaping cellular signaling. In the nervous system, growth factor-activated Trk receptor trafficking serves to convey biochemical signaling that underlies fundamental neural functions. Focus has been placed on axonal trafficking but little is known about growth factor-activated Trk dynamics in the neuronal soma, particularly at the molecular scale, due in large part to technical hurdles in observing individual growth factor-Trk complexes for long periods of time inside live cells. Quantum dots (QDs) are intensely fluorescent nanoparticles that have been used to study the dynamics of ligand-receptor complexes at the plasma membrane but the value of QDs for investigating ligand-receptor intracellular dynamics has not been well exploited. The current study establishes that QD conjugated brain-derived neurotrophic factor (QD-BDNF) binds to TrkB receptors with high specificity, activates TrkB downstream signaling, and allows single QD tracking capability for long recording durations deep within the soma of live neurons. QD-BDNF complexes undergo internalization, recycling, and intracellular trafficking in the neuronal soma. These trafficking events exhibit little time-synchrony and diverse heterogeneity in underlying dynamics that include phases of sustained rapid motor transport without pause as well as immobility of surprisingly long-lasting duration (several minutes). Moreover, the trajectories formed by dynamic individual BDNF complexes show no apparent end destination; BDNF complexes can be found meandering over long distances of several microns throughout the expanse of the neuronal soma in a circuitous fashion. The complex, heterogeneous nature of neuronal soma trafficking dynamics contrasts the reported linear nature of axonal transport data and calls for models that surpass our generally limited notions of nuclear-directed transport in the soma. QD-ligand probes are poised to provide understanding of how the molecular mechanisms underlying intracellular ligand-receptor trafficking shape cell signaling under conditions of both healthy and dysfunctional neurological disease models.
PLoS ONE 04/2014; 9(4):e95113. DOI:10.1371/journal.pone.0095113 · 3.23 Impact Factor
"Different types of peptides have been used as the promising candidates for intracellular delivery of QDs9101112131415. Recent advances include the enhancement of intracellular delivery of streptavidin-conjugated QDs into mouse fibroblast cells using biotinylated L-arginine peptides10, TAT-functionalized QDs for selective intracellular transport, vesicle shedding and delivery91314. Arginine–glycine–aspartic acid (RGD) peptides have been conjugated to target QDs specifically to tumor angiogenesis for theranostics1216. "
[Show abstract][Hide abstract] ABSTRACT: Protein transport is an important phenomenon in biological systems. Proteins are transported via several mechanisms to reach their destined compartment of cell for its complete function. One such mechanism is the microtubule mediated protein transport. Up to now, there are no reports on synthetic systems mimicking the biological protein transport mechanism. Here we report a highly efficient method of mimicking the microtubule mediated protein transport using newly designed biotinylated peptides encompassing a microtubule-associated sequence (MTAS) and a nuclear localization signaling (NLS) sequence, and their final conjugation with streptavidin-coated CdSe/ZnS quantum dots (QDs). Our results demonstrate that these novel bio-conjugated QDs enhance the endosomal escape and promote targeted delivery into the nucleus of human mesenchymal stem cells via microtubules. Mimicking the cellular transport mechanism in stem cells is highly desirable for diagnostics, targeting and therapeutic applications, opening up new avenues in the area of drug delivery.
"Thus, this study supported previous description of exosomes attaching to cell surface receptors (Miyanishi et al., 2007; Nolte-'t Hoen et al., 2009). Besides, the latter two intracellular modes indicated confined diffusion in local microenvironments of cytoplasm and active transport along cytoskeleton, highly correlated to the endocytic-derived transport reported for vesicles containing polyplexes (de Bruin et al., 2007), influenza virus (Lakadamyali et al., 2003), or golden nanoparticles (Ruan et al., 2007). The three-stage intracellular transport process of exosomes described before by us (Tian et al., 2010), similar to influenza transport (Lakadamyali et al., 2003), was also supported by this work. "
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