Enhanced Intracellular Delivery by Twisted CC-Loop Conformation in Cyclic Cell-Penetrating Peptides

Biological drugs hold great promise for treating various diseases, but their efficacy is often limited by poor cellular uptake. Herein, we introduce cyclic cell-penetrating peptides (CPPs) to enhance the delivery efficiency. Three cyclic peptides with varying ring sizes were designed from a classic amphiphilic CPP via disulfide bond formation. Among them, Y_2-13-OX adopted a twisted CC-loop conformation distinct from the typical β-hairpin on negatively charged cell surfaces. This unique structure enhanced membrane penetration, enabling superior delivery compared with linear and other cyclic variants. Y_2-13-OX efficiently delivered functional siRNA targeting METTL3, achieving knockdown comparable to that of Lipofectamine 2000. It also transported GFP and plasmids, demonstrating versatility. Computational analysis revealed molecular-level insights into the enhanced interaction between the CC-loop structure and membranes. These findings establish a new CPP conformation that advances therapeutic delivery and opens new avenues for drug transport strategies.