In contrast, penetratin-SNBD showed a punctate distribution indicative of vesicle accumulation (Figure ). Interestingly, these vesicles were not located in the membrane but internalized and distributed across the cytosol (Figures and), as confirmed by Z-stack imaging microscopy (Figure ). This observation correlates with the inverted micelle mechanism that was first described for penetratin. Interestingly, the co-administration of penetratin-SNBD with MiTMAB TM reduced the number of fluorescent intracellular vesicles (Figure ), suggesting that the internalization of penetratin can also partially occur via endocytosis as described in some literature reports. Lastly, the labeled peptide sC18-SNBD exhibited a typical endocytic mechanism of internalization (Figure ). After initial accumulation at the plasma membrane, sC18-SNBD showed clear intracellular punctate distribution (e.g., endosomes) and subsequently spread across the cells with high efficiency (Figure , Figure and Movie S3). In this case, the co-administration with MiTMAB TM did prevent the cell uptake to a large extent, thereby confirming that the internalization of sC18-SNBD was mediated by endocytosis (Figure ). The results of these experiments indicate that late-stage SNBD labeling is a straightforward strategy for non-perturbative modification of unprotected peptides and corroborate its application for mechanistic studies of internalization and trafficking in three prototypical peptide entities.