Cell penetrating peptides fail to induce an innate immune response in epithelial cells in vitro: implications for continued therapeutic use.

Cell penetrating peptides (CPPs) offer the exciting potential of effectively delivering macromolecules to the cytoplasm of a cell that are otherwise impermeable to the plasma membrane. Although the use of these peptides has so far been well tolerated in clinical trials, it is important to remember that some of these CPPs were originally derived from pathogenic material. We therefore sought to determine if three of the most widely studied CPPs; HIV-TAT, Antennapedia and Transportan, initiated an immune response in epithelial cells. Using conditions where these peptides efficiently delivered a rhodamine tagged BSA cargo to the interior of epithelial cells, we failed to observe an effect on cell viability as determined by MTT assay (P>0.05). Further, CPP-mediated delivery of this protein cargo failed to activate NFκB, which would be indicative of toll-like receptor signalling. Finally, no significant increase in the release of the inflammatory cytokines interleukin (IL)-8 and IL-6 was detected in epithelial cells exposed to CPP complexes for 72 h (P>0.05). Together, these results indicate that these commonly used CPPs are passive carriers that do not initiate epithelial cell-associated 'danger signals' during the process of cytoplasmic delivery of a model protein cargo.

Comparative study on transduction and toxicity of protein transduction domains.

BACKGROUND AND PURPOSE: Protein transduction domains (PTDs), such as Tat, antennapedia homeoprotein (Antp), Rev and VP22, have been extensively utilized for intracellular delivery of biologically active macromolecules in vitro and in vivo. There is little known, however, about the relative transduction efficacy, cytotoxicity and internalization mechanism of individual PTDs. EXPERIMENTAL APPROACH: We examined the cargo delivery efficacies of four major PTDs (Tat, Antp, Rev and VP22) and evaluated their toxicities and cell internalizing pathways in various cell lines. KEY RESULTS: The relative order of the transduction efficacy of these PTDs conjugated to fluorescein was Rev>Antp>Tat>VP22, independent of cell type (HeLa, HaCaT, A431, Jurkat, MOLT-4 and HL60 cells). Antp produced significant toxicity in HeLa and Jurkat cells, and Rev produced significant toxicity in Jurkat cells. Flow cytometric analysis demonstrated that the uptake of PTD-fluorescein conjugate was dose-dependently inhibited by methyl-beta-cyclodextrin, cytochalasin D and amiloride, indicating that all four PTDs were internalized by the macropinocytotic pathway. Accordingly, in cells co-treated with 'Tat-fused' endosome-disruptive HA2 peptides (HA2-Tat) and independent PTD-fluorescent protein conjugates, fluorescence spread throughout the cytosol, indicating that all four PTDs were internalized into the same vesicles as Tat. CONCLUSIONS AND IMPLICATIONS: These findings suggest that macropinocytosis-dependent internalization is a crucial step in PTD-mediated molecular transduction. From the viewpoint of developing effective and safe protein transduction technology, although Tat was the most versatile carrier among the peptides studied, PTDs should be selected based on their individual characteristics.