Membrane-Traversing Peptides: Mechanistic Landscapes, Assays, and Applications

Lipid bilayer membranes are essential cellular permeability barriers that strictly limit the passage of polar molecules and macromolecules, thereby compartmentalizing cellular biochemistry and limiting the accessible chemical space for therapeutics. Membrane-traversing peptides (MTPs) are a diverse group of peptides defined by the ability to traverse synthetic or cellular membranes without causing permeabilization and without the assistance of specific transport proteins. These peptides fundamentally defy classical thermodynamic models of membrane permeability as they can cross lipid bilayers and deliver large polar cargoes, including peptides and proteins, into cells, despite having high net charge and low hydrophobicity. In this review, we challenge the idea of single, discrete mechanisms to describe how peptides traverse membranes and define a broad mechanistic landscape shaped by the conserved properties of MTPs and the unique physical chemistry and polymorphic phase behavior of lipid bilayers. We describe experimental assays and model systems that can be used to study MTP translocation and cargo delivery in synthetic systems and living cells. We use information obtained from comprehensive databases to consider the shared physical chemical space of MTPs. We also discuss key unanswered questions regarding mechanism, specificity, endosomal escape, and in vivo performance. Finally, we discuss illustrative examples and emerging translational applications of MTPs in intracellular drug delivery and molecular therapeutics. Together, these perspectives underscore the dual importance of MTPs as probes for fundamental biophysical studies of cell membranes and as promising tools to expand the universe of druggable intracellular targets.