Quantitative Assessment of the Cell Penetrating Properties of RI-Tat-9:  Evidence for a Cell Type-Specific Barrier at the Plasma Membrane of Epithelial Cells

Abstract

Penetration of epithelial cells represents the rate-determining step for the absorption of many drugs and pharmaceutical macromolecules such as proteins and nucleic acid therapeutics. While the potential of using cell-penetrating peptides (CPPs) to facilitate absorption has been increasingly recognized, the mechanism of cell penetration and the uptake into certain cells have recently been called into question due to methodological artifacts. Therefore, the objective of this study was to quantitatively assess the ability of RI-Tat-9, a proteolytically stable CPP, to penetrate epithelial cell monolayers. The permeability of RI-Tat-9 with two epithelial cell lines, Madin-Darby canine kidney (MDCK) and Caco-2 cells, was comparable to the leakiness of the respective intact monolayers. Microscopic imaging showed that fluorescence-tagged RI-Tat-9 did not enter these cells, further supporting a paracellular transport mechanism. Although insufficient data were generated in these studies to generalize the observed phenomenon, the entry of RI-Tat-9 into nonepithelial T lymphocytic MT2 cells, possibly by endocytosis, suggested that a cell type-specific barrier might exist that controlled uptake of RI-Tat-9 by cells. Compared to that in MT2 and HeLa cells, the active uptake of the peptide into MDCK monolayers was much slower and showed no dependence of cell energy. Furthermore, the equilibrium binding of RI-Tat-9 to MDCK cells at 0 degrees C was indicative of an interaction with a nonspecific receptor. A correlation between binding density and concentration difference across a leaky separation barrier suggested that repulsion of free peptide molecules by bound peptide molecules at the MDCK monolayer surface may be significant at micromolar concentrations. The results of this study quantitatively show that Tat CPP uptake into two commonly used epithelial cell types is minimal and possibly cell type-specific. Implications for Tat CPP-assisted drug delivery are discussed.

Identification of a Signal Peptide for Unconventional Secretion

“…Quantitative analysis of SecPen transfer from ten independent experiments indicates that, following the addition of 10 nmol of peptide in the basolateral compartment, 3.3 Ϯ 0.7 pmol of peptide are retrieved in the apical compartment after 1 h. In similar conditions, the transcytosis of transferrin is 50-fold less efficient. Previous studies on the passage of PTDs across epithelial monolayers have demonstrated that Tat and Penetratin PTDs are incapable of transcellular transport (13)(14)(15)(16) and, moreover, are poorly internalized by confluent MDCK cells (13,14,16). In agreement with these observations, we found that neither of these two peptides is transported across the epithelium in our system and that Penetratin internalization is significantly reduced when MDCK cells reach confluence (not shown).…”

Differentiation Restricted Endocytosis of Cell Penetrating Peptides in MDCK Cells Corresponds with Activities of Rho-GTPases

“…In one of our previous studies to this subject, using linear human calcitonin derived CPPs, Tat (47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57) and penetratin (43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58), internalization into proliferating HeLa cells was superior to that in a fully polarized and differentiated MDCK model (7). Similarly, Zhang et al (24) suggested a cell type-specific barrier to control the internalization of RI-Tat-9 into HeLa, MT2, MDCK and Caco-2 cells.…”

“…Cell line dependence of CPP internalization has been suggested by different research groups (7,23,24). Previously, Hallbrink et al (34) hypothesized about a cell density dependent internalization of CPPs.…”

“…In the majority of cases, Bleaky^cell models, such as HeLa, KB 3-1, Bowes Human Melanoma or MC57 fibrosarcoma cells, have been used for CPP internalization (19)(20)(21)(22)(23). When cultured on solid surfaces such cell lines lack the ability to form tight junctions and form cell layers of high permeability (24). Cellular models with junctional complexes bear more relevance for therapeutic considerations, namely for the delivery of CPP-cargo constructs into epithelia.…”

Cell-penetrating peptides as delivery vehicles for biology and medicine

“…drug, fluorophore, nanoparticle, protein) and on cell type. 67 Moreover, uptake mechanism can be misassigned as a consequence of experimental artifacts, such as those resulting from cell fixation or omission of a proteolytic digestion. 68 In addition, some pharmacological agents used to probe specific mechanisms of endocytosis can interfere with multiple endocytic pathways as well as other cellular properties.…”