Peptide-Based Strategies of Delivering Semiconductor Nanocrystals into Living Cells
thesisposted on 08.02.2018, 00:00 by Claudia Ivette Rivera Vera
Semiconductor nanocrystals or quantum dots (QDs) possess exceptional optical and physicochemical properties, for instance, their brightness and long fluorescence lifetimes, which makes them valuable for live-cell imaging. Due to their large size, QDs do not cross cell membranes passively, fortunately, this issue can be addressed by coating QDs with cell penetrating peptides (CPPs) such as the TAT sequence or polyarginine as it has been reported by several studies from different labs. We could intracellularly deliver CPP coated red and green emitting QDs, however, as many others have shown, we also found that endocytosis was the major mechanism of intracellular uptake in HeLa cells, and consequently, QDs remained trapped inside the endosomes without being able to reach the cytosol. In this thesis, I designed and synthesized a few peptides with purported potential endosomal escape activity. These peptides were conjugated to the QD surface, and we showed that a peptide derived from Aurein 1.2 and a polyamine derivative significantly enhanced the endosomal escape of our QD bioconjugates in live cell studies. Further, we developed a FRET (Forster resonance energy transfer)-based method to quantify the endosomal escape triggered by the Aurein 1.2, polyamine, and palmitoyl peptides respectively, along with a small protein (HA2) derived from the influenza virus. Our FRET sensor system is comprised of a QD (as the FRET donor) linked to a rhodamine labeled peptide (as the FRET acceptor) through a disulfide bond, and thus, rendering a ratiometric FRET system that measures the extent of endosomal escape by the change in red/green signal upon disulfide bond cleavage by cytosolic glutathione. Collectively, our qualitative and quantitative microscopy studies indicate that both, Aurein 1.2, as well as a polyamine peptide I designed from scratch, do enhance endosomal escape of QDs in live-cell studies. I also performed more than 95% of the synthetic work displayed in this thesis.