Cell-penetrating activity of a short-chain ε-poly-l-α-lysine

Bacteria produce polycationic homopoly(amino acid)s, which are characterized by isopeptide backbones. We previously demonstrated that two representative bacterial polycationic isopeptides, ε-poly-l-α-lysine consisting of 25–35 l-α-lysine residues (ε-PαL_25-35) and ε-poly-l-β-lysine consisting of l-β-lysine residues (ε-PβL_4-13), were internalized into mammalian cells by both energy-independent direct penetration and energy-dependent endocytosis/macropinocytosis, and then diffused throughout the cytosol. In this study, we investigated the cell-penetrating activity of an ε-PαL short-chain derivative consisting of 5–14 l-α-lysine residues (ε-PαL_5-14) to gain insight into the relationship between the isopeptide-chain length and the manner of cellular internalization. We prepared a conjugate of ε-PαL_5-14 and a fluorescent dye (FAM) by click chemistry, and incubated the resulting polymer, ε-PαL_5-14-FAM, with HeLa cells. Unlike ε-PαL_25-35-FAM, ε-PαL_5-14-FAM was internalized into cells only by energy-dependent endocytosis/macropinocytosis. Furthermore, a high concentration (>50 μM) was required for the internalization events. ε-PαL_5-14 has a chain length almost equal to that of the membrane permeable ε-PβL_4-13, which can enter cells at low concentrations. Considering that the basicity of the β-amino group is higher than that of α-amino acid at physiological pH, ε-PβL is expected to have a greater cell-penetrating capacity than ε-PαL, provided their isopeptide-chain lengths are similar, suggesting that a more extended chain derivative of ε-PβL would be more advantageous for cellular internalization of cargo proteins than ε-PαL_25-35.