Mechanistic Insights into Unary Peptide–Membrane Interactions Enable Stable Encapsulation and Trigger-Responsive Peptidyl Liposomes

Efforts to engineer trigger-responsive peptidyl liposomes have historically been limited by premature release following peptide conjugation, reflecting an incomplete understanding of tethered peptide-membrane interactions. Here, we establish a unified mechanistic framework for designing encapsulation-stable yet trigger-responsive liposomes by elucidating how membrane-anchored peptides interact with membranes. Screening identified MAG2 as, thus far, the only AMP backbone that can be surface-masked without liposome leakage while preserving latent lytic activity─-an outcome previously unattainable in unary peptidyl liposome systems. Cryo-EM/cryo-ET, SAXS, CD, FLIM/fluorescence imaging, and MTT assays collectively unveil a hierarchical cascade of molecular events: masked peptide-membrane conjugation with stable liposome encapsulation, lateral-diffusion-driven outer leaflet PEG-layer expansion, trigger-induced peptide unmasking, aggregation-mediated membrane defect formation, and outer leaflet PEG-layer collapse, culminating in liposomal content release, intracellular distribution, and trigger-induced cell-killing efficacy. These findings uncover a unary peptide-membrane interaction mechanism that will redefine the design principles of trigger-responsive therapeutic peptidyl liposomes.