Triggered cargo release from liposomes using membrane-selective nanopore-forming peptides
Here we developed a model of peptide-triggered cargo release from liposomes using peptides that have extraordinary selectivity for PC liposomes over mammalian cells. Such a model can be used when cargoes are encapsulated into stable liposomes, which can be permeabilized by peptides so that the cargoes are released immediately in the presence of cells. Previously, a novel family of α-helical pore-forming peptides called "macrolittins", was identified. These 26-residue peptides are products from two batches of screening iterative library. Macrolittins bind strongly to phosphatidylcholine (PC) liposomes, fold into α-helical secondary structure, and form macromolecule-sized pores, releasing macromolecular cargoes from PC liposomes at concentrations as low as 1 peptide per 1000 lipids. In this work, we show that macrolittins bind very weakly to cell membranes and have no measurable cytolytic activity against multiple human cell types even at a high peptide concentration. We show that this unprecedented selectivity for PC liposomes over cell plasma membranes can be explained, in part, by the sensitivity of macrolittin activity to physical chemical properties of the bilayer hydrocarbon core. We also find that macrolittins can cause PC liposome aggregation and fusion as well as increased fusion between PC liposome and cell membrane, but that these activities can be inhibited by inclusion of pegylated phospholipid within the membrane without affecting permeabilization. In the presence of cells, macrolittins release all liposome-entrapped cargoes (proteins and small molecule drugs) which are then readily uptaken by the cells. Triggered release occurs without any direct effect of the peptides on the cells, and without vesicle-vesicle or vesicle-cell interactions. To further investigate sequence-structure-function relationship of pore forming peptides, we made a series of targeted substitutions and demonstrated the importance of two highly conserved acidic amino acids at the 4th and 8th positions in the macrolittin sequence. Finally, to further explore the basis for membrane selectivity, a library of elongated macrolittin analogues was synthesized and screened against liposomes of different membrane thicknesses. We find that variant hits have longer α-helical secondary structure, but have a preserved helical pattern of charged residues, which are required to partition into and form nanopores in thicker bilayers.