Chemical Synthesis of Mirror-Image Proteins Reveals Chirality-Dependent Cellular Uptake

Mirror-image peptides and proteins are attracting interest as therapeutics, key building blocks for constructing mirror-image life, and as tools to probe the origin of life. Their resistance to proteolytic degradation and unique stereochemistry make D-peptides/proteins particularly appealing for biomedical applications, yet a critical unresolved question is how their intracellular uptake compares with that of natural L-forms. To address this, we systematically investigated the role of cargo chirality in cellular internalization while maintaining a constant delivery vehicle. Three model cargos of increasing size and structural complexity were synthesized in both L- and D-configurations and conjugated to an identical cyclic deca-arginine (cR10) cell-penetrating peptide (CPP). By keeping the CPP scaffold constant, we reduced delivery-related variability and directly assessed the influence of cargo chirality on uptake. Quantitative uptake analysis using flow cytometry, gel analysis, and confocal microscopy across multiple mammalian cell lines reveals that L-cargos are internalized more efficiently than their mirror-image D-counterparts, demonstrating that cargo chirality is a key determinant of uptake efficiency across the chiral biological membrane. Collectively, these findings provide a systematic basis for further exploration of chirality effects in CPP-mediated delivery and may inform the design of mirror-image peptides and proteins for therapeutic or synthetic biology applications.