Endocytosis and Membrane Potential Are Required for HeLa Cell Uptake of R.I.-CKTat9, a Retro-Inverso Tat Cell Penetrating Peptide

Abstract

Cell-penetrating peptides (CPPs) can enter many types of cells and have become useful tools for introducing a variety of cargo such as exogenous peptides, proteins, and nucleic acids into cultured cells in vitro. Tat CPPs derived from the HIV-1 Tat protein are the most widely used among the arginine-rich CPPs. Even though CPPs hold considerable promise for drug delivery, poor biological stability and high in vivo clearance may limit their effectiveness for delivering cargo. Therefore, we utilize a retro-inverso form of a Tat peptide, R.I.-CKTat9, which is proteolytically stable. In the current study, the cellular entry mechanism of this arginine-rich CPP is investigated. Fluorescently labeled R.I.-CKTat9 entered HeLa cells in a concentration-and energy-dependent manner demonstrating both diffuse and punctate (vesicular) appearance inside the cells. The labeled R.I.-CKTat9 colocalized with labeled transferrin in the punctate structure, suggesting that the peptide enters HeLa cells by clathrin-dependent endocytosis. Incubation of cells with an isotonic/high K + buffer (KPBS) or an NH 4 Cl solution abolished the diffuse but not the punctate fluorescence, suggesting that membrane potential plays a critical role. This result also suggests that the flux originates from the endosome, not the extracellular space, and relies on the acidity of the endosome. Impairment of clathrin-mediated endocytosis by RNAi with clathrin heavy chain function and endocytosis inhibitors greatly reduced or completely abolished both diffuse and punctate fluorescence, further supporting a single route of endocytosis and subsequent endosomal escape. Since cells in the mitotic (M) phase shut down endocytosis but maintain plasma membrane potential, this property was used to further confirm the endocytic mechanism. Direct measurement of plasma membrane potential confirmed its persistence in M phase arrested HeLa cells. Consistent with our working hypothesis, these cells did not show any vesicular nor diffuse fluorescence of labeled R.I.-CKTat9, providing compelling evidence for the sequential steps of endocytosis and endosomal escape. Binding of labeled R.I.-CKTat9 to the surface of HeLa cells at 0 °C was reduced under the mildly acidic conditions of early endosomes, suggesting an aciditydependent endosomal escape mechanism. Overall, these results indicate that both endocytosis and membrane potential are required for R.I.-CKTat9 entry into HeLa cells and suggest that translocation occurs at the endosomal membrane.

Direct cytosolic delivery of cargoes in vivo by a chimera consisting of D- and L-arginine residues

“…This indicates that the major internalization pathway of (rR) 3 R 2 is not endocytosis but more likely direct membrane translocation, another possible entry route of CPPs. Direct membrane translocation has been proposed to lead to the diffuse distribution of CPPs in the cytosol and nucleus [25,27,56] and some groups believed it was driven by the transmembrane potential [26,[59][60][61]. Thus, we investigated whether the membrane potential affected the cellular uptake of (rR) 3 R 2 .…”

Surface modifications of nanocarriers for effective intracellular delivery of anti-HIV drugs

“…The specificity of intracellular delivery is related to many factors including the type and number of targeting ligands required for optimal cellular uptake. Intracellular disposition and fate are highly dependent on the type of cell surface receptor and may require an additional strategy to promote endosomal escape [39]. These specific interactions result in preferential accumulation of nanocarrier systems into cellular targets [40].…”

“…Biodegradation inside the target cells has been conceptually attempted in this type of nanocarrier [66]. Tat cell penetrating peptide has been used as surface modifier to facilitate endosomal escape [39]. …”

Calcium Condensation of DNA Complexed with Cell-Penetrating Peptides Offers Efficient, Noncytotoxic Gene Delivery

“…Cell-penetrating peptides (CPPs) offer a potential alternative to PEI. These short polycations achieve intracellular access by crossing the plasma membrane directly or by endocytosis 19–25 while typically exhibiting low cytotoxicity 26,27 . Covalently conjugating CPPs to gene vectors (e.g.…”