The role of endocytosis on the uptake kinetics of luciferin-conjugated cell-penetrating peptides

Abstract

Cell-penetrating peptides (CPPs) are short cationic/amphipathic peptides that can be used to deliver a variety of cargos into cells. However, it is still debated which routes CPPs employ to gain access to intracellular compartments. To assess this, most previously conducted studies have relied on information which is gained by using fluorescently labeled CPPs. More relevant information whether the internalized conjugates are biologically available has been gathered using end-point assays with biological readouts. Uptake kinetic studies have shed even more light on the matter because the arbitrary choice of end-point might have profound effect how the results could be interpreted. To elucidate uptake mechanisms of CPPs, here we have used a bioluminescence based assay to measure cytosolic delivery kinetics of luciferin-CPP conjugates in the presence of endocytosis inhibitors. The results suggest that these conjugates are delivered into cytosol mainly via macropinocytosis; clathrin-mediated endocytosis and caveolae/lipid raft dependent endocytosis are involved in a smaller extent. Furthermore, we demonstrate how the involved endocytic routes and internalization kinetic profiles can depend on conjugate concentration in case of certain peptides, but not in case of others. The employed internalization route, however, likely dictates the intracellular fate and subsequent trafficking of internalized ligands, therefore emphasizing the importance of our novel findings for delivery vector development.

Overexpression of atypical protein kinase C in HeLa cells facilitates macropinocytosis via Src activation

“…Because macropinocytosis has been implicated in the internalization of some cell penetrating peptides that are rich in arginine or lysine [42–44] and the large size of the peptide-labeled vacuoles, we asked if the aPKC-PS peptide induced structures are macropinosomes by analyzing the uptake of a fluid phase tracer. For these studies, HeLa cells plated on coverslips were incubated with aPKC-PS peptide or with aPKC-SC peptide in the presence of 70 kDa Texas red-dextran (TXrDex) for 15 min at 37°C.…”

Gemcitabine upregulates ABCG2/BCRP and modulates the intracellular pharmacokinetic profiles of bioluminescence in pancreatic cancer cells

“…Examples include using a bioluminescence-based assay to measure cytosolic delivery kinetics of luciferin-cellpenetrating peptides conjugates in the presence of endocytosis inhibitors to elucidate uptake mechanisms of cell-penetrating peptides [24,25]. Furthermore, other reports explored whether the observed luminescence was due to intracellular release of luciferin or from a side reaction with luciferase.…”

Direct Cytosolic Delivery of Polar Cargo to Cells by Spontaneous Membrane-translocating Peptides

“…Since the discovery and characterization of the cell penetrating capabilities of the cationic Tat sequence more than 20 years ago, there has been excitement about the possibilities for the use of CPPs as a generic delivery platform for polar molecules (10,12,13,15,20,26,40). Accordingly, there has been a significant amount of work done to engineer and modify the known CPPs to improve their utility and to explore the mechanistic principles of cell penetration.…”