Imaging and Tracking of Tat Peptide-Conjugated Quantum Dots in Living Cells:  New Insights into Nanoparticle Uptake, Intracellular Transport, and Vesicle Shedding

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.

The performance of gradient alloy quantum dots in cell labeling

“…2A). The data reveal a clear colocalization of either QDot500 or QDot600 with the fluorescent lysosomes, indicating that both types of QDots were taken up by the cells through the process of endocytosis, as is the case for most types of NPs [34]. The gradient alloy QDots thus appear to finally reside in the lysosomal compartment of the cell, where they will be exposed to the low pH as described above.…”

“…Small NPs such as QDots are typically endocytosed by cultured cells [34], which results in their enclosure in endosomes and in a later phase, lysosomes, which are degradative cellular organelles that have a low pH (5.5 for endosomes, 4.5 for lysosomes). This low pH can affect the QDots by degrading their organic surface coating [35], following by acid etching of the QDot surface, resulting in a reduced PLQY and release of Zn2+ and Cd2+ ions [19,22].…”

“…As the uptake mechanism and final intracellular localization of NPs may differ depending on the size of the NPs and their specific surface chemistry [34], the cellular uptake of the QDots was investigated.…”

Microscopic observation of the intercellular transport of CdTe quantum dot aggregates through tunneling-nanotubes

“…The internalized QDs were not diffusely distributed inside the cell. A common observation is that QDs tend to aggregate inside living cells and are often trapped in organelles such as vesicles, endosomes, and lysosomes [29,30]. Microscopic analysis further revealed that the internalized QD aggregates could be transported along TNTs.…”

In vitro transfection of bone marrow-derived dendritic cells with TATp-liposomes

“…Clearly, the TATp-L transfected DC more efficiently because of their specific binding to the cell surface 48 and their ability to escape from endosomes by destabilizing them. 39,[49][50][51][52][53][54] These experiments with EGFP demonstrate the possibility of efficient DC transfection. To prove that the expressed protein can be immunologically active, we repeated some experiments using pCIgD A instead of pEGFP.…”