pH-Responsive Peptide\nNanoparticles Deliver Macromolecules\nto Cells via Endosomal Membrane Nanoporation

The synthetically evolved pHD family of peptides is known\nto self-assemble\ninto macromolecule-sized nanopores of 2–10 nm diameter in synthetic\nlipid bilayers, but only when the pH is below ∼6. Here, we\nshow that a representative family member, pHD108, has the same pH-responsive\nnanopore-forming activity in the endosomal membranes of living human\ncells, which is triggered by endosomal acidification. This enables\nthe cytosolic delivery of endocytosed proteins and other macromolecules.\nAcylation of either peptide terminus significantly decreases the concentration\nof peptide required for macromolecule delivery to the cell cytosol\nwhile not causing any measurable cytotoxicity. Longer acyl chains\nare more effective. The N-terminal palmitoylated C16-pHD108 is the\nmost potent of all of the acyl-pHD108 variants and readily delivers\na cytotoxic enzyme, fluorescent proteins, and a dye-labeled dextran\nto the cell cytosol. C16-pHD108 forms stable monodisperse micellar\nnanoparticles in a buffer at pH 7 with an average diameter of around\n120 nm. These nanoparticles are not cytolytic or cytotoxic because\nthe acylated pHD peptide does not partition from the nanoparticles\ninto cell membranes at pH 7. At pH 5, the nanoparticles are unstable,\ndriving acylated pHD108 to bind strongly to membranes. We hypothesize\nthat passive endocytosis of macromolecular cargo and stable peptide\nnanoparticles, followed by endosomal acidification-dependent destabilization\nof the nanoparticles, triggers the nanopore-forming activity of acylated\npHD peptides in the endosomal membrane, enabling internalized macromolecules\nto be delivered to the cytosol.