Life has evolved the plasma membrane as an active physicochemical interface rather than a passive barrier, one whose selectivity sustains homeostasis but excludes many therapeutic molecules. Yet most delivery strategies force molecules across this boundary, leaving the interface underused as a surface for organizing transport. Inspired by membrane-associated phase separation during endocytosis, we show that confining phase separation to the plasma-membrane interface lets cell-penetrating peptide (CPP) and silk fibroin (SF) co-condense in situ, coupling cargo recruitment, membrane wetting and entry into one continuous step. CPP-to-SF stoichiometry tunes the condensate between liquid- and solid-like states, determining whether it crosses the membrane or merely coats it. Cryo-TEM, STED, FRAP, live-cell tracking and in situ cryo-FIB/cryo-TEM resolved the sequence from membrane nucleation within 30 s to intracellular entry within 5 min, orders of magnitude faster than previous phase-separation-based delivery. Set by phase state rather than cargo identity, the system delivered small molecules, nucleic acids and antibodies at roughly 10-fold lower CPP dose. By acquiring cytoplasmic access after entry and then re-exiting cells, the same interfacial condensates supported transcellular traversal across the intact cornea in vivo, lowering intraocular pressure with a 4.2-fold lower betaxolol dose and delivering otherwise excluded siRNA into the anterior chamber. More broadly, this establishes the membrane interface as a programmable determinant of barrier permeability, extending beyond this system to other condensate-forming partners and barriers.
Han, L., Wan, Y., Guo, Z., Gao, S., Wang, N., Mao, Y.
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