Cell-Penetrating Peptides in Diagnosis and Treatment of Human Diseases: From Preclinical Research to Clinical Application

The presence of cell and tissue barriers together with the low biomembrane permeability of various therapeutics often hampers systemic drug distribution; thus, most of the available molecules are of limited therapeutic value. Opportunities to increase medicament concentrations in areas that are difficult to access now exist with the advent of cell-penetrating peptides (CPPs), which can transport into the cell a wide variety of biologically active conjugates (cargoes). Numerous preclinical evaluations with CPP-derived therapeutics have provided promising results in various disease models that, in some cases, prompted clinical trials. The outcome of these investigations has thus opened new perspectives for CPP application in the development of unprecedented human therapies that are well tolerated and directed to intracellular targets.

The TAT protein transduction domain (PTD) has been used to deliver a wide variety of biologically active cargo for the treatment of multiple preclinical disease models, including cancer and stroke. However, the mechanism of transduction remains unknown. Because of the TAT PTD's strong cell-surface binding, early assumptions regarding cellular uptake suggested a direct penetration mechanism across the lipid bilayer by a temperature- and energy-independent process. Here we show, using a transducible TAT-Cre recombinase reporter assay on live cells, that after an initial ionic cell-surface interaction, TAT-fusion proteins are rapidly internalized by lipid raft-dependent macropinocytosis. Transduction was independent of interleukin-2 receptor/raft-, caveolar- and clathrin-mediated endocytosis and phagocytosis. Using this information, we developed a transducible, pH-sensitive, fusogenic dTAT-HA2 peptide that markedly enhanced TAT-Cre escape from macropinosomes. Taken together, these observations provide a scientific basis for the development of new, biologically active, transducible therapeutic molecules.

A basic peptide derived from human immunodeficiency virus (HIV)-1 Tat protein (positions 48-60) has been reported to have the ability to translocate through the cell membranes and accumulate in the nucleus, the characteristics of which are utilized for the delivery of exogenous proteins into cells. Based on the fluorescence microscopic observations of mouse macrophage RAW264.7 cells, we found that various arginine-rich peptides have a translocation activity very similar to Tat-(48-60). These included such peptides as the d-amino acid- and arginine-substituted Tat-(48-60), the RNA-binding peptides derived from virus proteins, such as HIV-1 Rev, and flock house virus coat proteins, and the DNA binding segments of leucine zipper proteins, such as cancer-related proteins c-Fos and c-Jun, and the yeast transcription factor GCN4. These segments have no specific primary and secondary structures in common except that they have several arginine residues in the sequences. Moreover, these peptides were able to be internalized even at 4 degrees C. These results strongly suggested the possible existence of a common internalization mechanism ubiquitous to arginine-rich peptides, which is not explained by a typical endocytosis. Using (Arg)(n) (n = 4-16) peptides, we also demonstrated that there would be an optimal number of arginine residues (n approximately 8) for the efficient translocation.