cR10 cell-penetrating peptide delivers L-cargos more efficiently than D-mirror-image counterparts into mammalian cells

D-peptides and D-proteins offer therapeutic promise due to their enhanced proteolytic resistance and unique stereochemistry, making them attractive for drug development. However, a critical gap exists in understanding how their cellular internalization compares to natural L-forms. Efficient intracellular delivery is paramount for therapeutic efficacy, yet the influence of cargo chirality on cell-penetrating peptide (CPP)-mediated uptake across biological membranes remains largely unexplored. This study directly investigates this fundamental question to inform future therapeutic design.

Researchers synthesized three model cargos of varying size and complexity in both L- and D-configurations. These cargos were then conjugated to an identical cyclic deca-arginine (cR10) cell-penetrating peptide to ensure a constant delivery vehicle. The study systematically investigated the cellular internalization of these constructs across multiple mammalian cell lines. Quantitative uptake analysis was performed using flow cytometry, gel analysis, and confocal microscopy to directly assess the influence of cargo chirality on uptake efficiency.

Quantitative uptake analysis consistently revealed that L-cargos were internalized more efficiently than their mirror-image D-counterparts. This observation was consistent across all three model cargos, irrespective of their size or structural complexity, and was confirmed across multiple mammalian cell lines. The findings demonstrate that cargo chirality is a critical determinant of uptake efficiency during CPP-mediated delivery. The study highlights that even with an identical cR10 CPP, the stereochemistry of the cargo significantly impacts its ability to traverse the chiral biological membrane. This systematic investigation provides a foundational understanding of chirality effects in cellular internalization.