Cell-penetrating peptides are short sequences of amino acids capable of inserting into cell membranes. The amphipathic peptide R6W3 (RRWWRRWRR) has previously been shown to enhance endocytic activity in cells. Here, we demonstrate that R6W3 can also drive direct translocation into lipid vesicles that lack endocytic machinery. Specifically, R6W3 facilitates transport of fluorescent dye into vesicle lumens, but only when the dye is covalently attached to the peptide. In contrast, co-incubation of free dye with R6W3 leaves the dye excluded from the vesicle lumen. These findings suggest that R6W3 undergoes translocation without causing poration of the bilayer. Vesicle uptake is dye mediated and light responsive, increasing in a dose-dependent manner with fluorescence excitation. Remarkably, peptide-mediated dye accumulation occurs in a wide range of synthetic lipid vesicles and in cell-derived giant plasma membrane vesicles, with fluorescence intensities in the lumen reaching up to 100× those of the external solution. This work establishes a new route for optical-based vesicle loading and highlights a potential mechanism for cellular delivery that bypasses endocytosis.
Werb et al. (Sun,) studied this question.