Amphipathic cell-penetrating peptide-based molecular transporters: A balanced approach for membrane penetration

The intracellular delivery of therapeutic biomacromolecules remains a fundamental challenge in modern drug development, primarily because the plasma membrane is inherently impermeable to large, hydrophilic molecules. While conventional small-molecule drugs also suffer from short circulation half-lives and off-target interactions, biologics face an even more critical barrier: the inability to efficiently access cellular targets. Cell-penetrating peptides (CPPs), capable of traversing lipid bilayers, have emerged as powerful intracellular delivery vectors, offering structural tunability, ease of chemical modification, and broad cargo compatibility. Among the diverse CPP classes, amphipathic CPPs occupy a uniquely advantageous position, balancing hydrophobic and hydrophilic domains to enable efficient membrane penetration while maintaining lower cytotoxicity and superior aqueous solubility compared with purely cationic or hydrophobic counterparts. This review provides a comprehensive and critical analysis of amphipathic CPPs, encompassing their structural classification and key cellular internalization pathways. Particular attention is devoted to rational design principles governing transporter performance, including the strategic combination of arginine and tryptophan residues, disulfide-mediated redox-responsive cyclization, fatty acid conjugation, and the progressive advancement in polycyclization to enhance proteolytic stability and cellular uptake. Emerging applications spanning cancer therapy, antimicrobial strategies, gene editing, nucleic acid delivery, and biosensing are also surveyed. Finally, current limitations, including endosomal sequestration, limited mechanistic understanding, and inconsistent structure-uptake relationships, are critically evaluated alongside future directions, including artificial intelligence and machine learning-driven peptide design and high-throughput screening approaches. Overall, the insights documented aim to guide peptide chemists in the rational design of next-generation amphipathic CPP-based molecular transporters with genuine clinical translation potential.