Cellular Trafficking and Photochemical Internalization of Cell Penetrating Peptide Linked Cargo Proteins: A Dual Fluorescent Labeling Study

摘要

Initial cellular uptake of cell penetrating peptide (CPP) linked macro-molecules is usually endosomal, with passage from endosome to cytosol a major limitation to efficient delivery. To gain a better understanding of the passage of the CPP-linked proteins, we studied the uptake and localization of CPP-linked proteins that contained two different forms of fluorescent markers, GFP protein and chemically conjugated tetra-methylrhodamine, in living cells. Rhodamine labeled TAT-GFP was internalized in multiple cell lines including HEK293, N18-RE-105, hippocampal slices, and human neural progenitor cells and showed predominandy endosomal localization of both fluorescent markers. Cytosolic localization of some rhodamine label was detected to suggest that some of the GFP label had exited from the endosome. However, quantification of the distribution of the rhodamine and GFP label indicated that the protein location was primarily endosomal and that the distribution of TAT-GFP was not significandy different than that of an exclusively endosomal localized exogenous protein (tetanus toxin fragment C- TTC). As a result, photochemical internalization (PCI) was evaluated and caused a significant Quantitative redistribution of cellular fluorescence of rhodamine anr caused a significant quantitative redistribution of cellular fluorescence of rhodamine and GFP labels to demonstrate increased cytosolic delivery of GFP. While rhodaniine-labeled TAT-GFP showed cytosolic delivery with exposure to specific wavelengths of fluorescent illumination, a similarly labeled GFP fusion protein containing the membrane binding domain of TTC did not mediate PCI in N18-RE-105 cells. .