Direct Cytosolic Uptake of Cell Penetrating Peptides with Shortened Sidechains

Endocytosis and direct translocation serve as the primary mechanisms by which cell‐penetrating peptides (CPPs) enter cells. Understanding these pathways is vital for unraveling the complexities of cellular uptake, which bears significant relevance to progress in various fields. In this study, the intracellular distribution of arginine octamer analogs is demonstrated to correlate with the length of their side chains. Specifically, peptides with elongated sidechains exhibit enhanced cellular uptake efficiency and a preferential endosomal distribution, whereas mR8—a peptide with a shortened sidechain—primarily enters cells via direct translocation and displays cytosolic/nuclear localization. It is further revealed that these structurally analogous peptides exhibit distinct diffusion characteristics and complexation behaviors with model glycosaminoglycans. These findings contribute to a deeper molecular understanding of the cellular uptake mechanisms of peptides.