Enhanced liposomal drug delivery via membrane fusion triggered by dimeric coiled-coil peptides

An ideal nanomedicine system improves the therapeutic efficacy of a drug. However, most nanomedicines enter the cell via endosomal/lysosomal pathways and typically only a small fraction of the cargo enters the cytosol inducing a therapeutic effect. To circumvent these inefficient drug delivery pathways, alternative approaches are desired. Inspired by fusion machinery found in nature, various synthetic fusogens have been developed to mediate the fusion of vesicles. Previously we used lipidated versions of the heterodimeric peptide pair E4/K4 to induce membrane fusion and applied this approach to deliver drugs in vitro and in vivo. Peptide K4 was designed to interact specifically with peptide E, but it was also shown to have an affinity for lipid membranes resulting in membrane remodeling. As the next step towards efficient in vivo membrane fusion for potential therapeutic applications, our rationale was to design efficient fusogen variants that could have multiple interactions simultaneously. To achieve this goal, we synthesized dimeric coiled-coil peptide variants of peptide K to facilitate improved fusion with peptide E-modified liposomes and mammalian cells. The secondary structure and self-assembly of various dimers were studied; the parallel PK4 dimer forms temperature-dependent higher-order assemblies, while linear K4 dimers form tetramer-like homodimers. The findings regarding the structures and membrane interactions of the PK4 dimer were supported by molecular dynamics simulations. Upon addition of the complementary peptide E4, the parallel PK4 dimer induced the strongest coiled-coil interaction resulting in a higher cellular uptake of the liposome-encapsulated cargo, as compared to the linear dimer and monomeric designs. Using a wide spectrum of endocytosis inhibitors, it was shown that membrane fusion was the main cellular uptake pathway. Delivery of the antitumor drug doxorubicin (DOX) resulted in efficient cellular delivery and concomitant antitumor efficacy. These findings not only offer important mechanistic insights into the design of coiled-coil driven membrane fusion systems but also provide novel strategies for developing peptide-based biomaterials.