Delivering quantum dot-peptide bioconjugates to the cellular cytosol: escaping from the endolysosomal system

For luminescent quantum dots (QDs) to realize their full potential as intracellular labeling, imaging and sensing reagents, robust noninvasive methods for their delivery to the cellular cytosol must be developed. Our aim in this study was to explore a range of methods aimed at delivering QDs to the cytosol. We have previously shown that QDs functionalized with a polyarginine 'Tat' cell-penetrating peptide (CPP) could be specifically delivered to cells via endocytic uptake with no adverse effects on cellular proliferation. We began by assessing the long-term intracellular fate and stability of these QD-peptide conjugates. We found that the QDs remained sequestered within acidic endolysosomal vesicles for at least three days after initial uptake while the CPP appeared to remain stably associated with the QD throughout this time. We next explored techniques designed to either actively deliver QDs directly to the cytosol or to combine endocytosis with subsequent endosomal escape to the cytosol in several eukaryotic cell lines. Active delivery methods such as electroporation and nucleofection delivered only modest amounts of QDs to the cytosol as aggregates. Delivery of QDs using a variety of transfection polymers also resulted in primarily endosomal sequestration of QDs. However, in one case the commercial PULSin reagent did facilitate a modest cytosolic dispersal of QDs, but only after several days in culture and with significant polymer-induced cytotoxicity. Finally, we demonstrated that an amphiphilic peptide designed to mediate cell penetration and vesicle membrane interactions could mediate rapid QD uptake by endocytosis followed by a slower efficient endosomal release which peaked at 48 h after initial delivery. Importantly, this QD-peptide bioconjugate elicited minimal cytotoxicity in the cell lines tested.