pH-Responsive Peptide Nanoparticles Deliver Macromolecules to Cells via Endosomal Membrane Nanoporation.

The synthetically evolved pHD family of peptides is known to self-assemble into macromolecule-sized nanopores of 2-10 nm diameter in synthetic lipid bilayers, but only when the pH is below ∼6. Here, we show that a representative family member, pHD108, has the same pH-responsive nanopore-forming activity in the endosomal membranes of living human cells, which is triggered by endosomal acidification. This enables the cytosolic delivery of endocytosed proteins and other macromolecules. Acylation of either peptide terminus significantly decreases the concentration of peptide required for macromolecule delivery to the cell cytosol while not causing any measurable cytotoxicity. Longer acyl chains are more effective. The N-terminal palmitoylated C16-pHD108 is the most potent of all of the acyl-pHD108 variants and readily delivers a cytotoxic enzyme, fluorescent proteins, and a dye-labeled dextran to the cell cytosol. C16-pHD108 forms stable monodisperse micellar nanoparticles in a buffer at pH 7 with an average diameter of around 120 nm. These nanoparticles are not cytolytic or cytotoxic because the acylated pHD peptide does not partition from the nanoparticles into cell membranes at pH 7. At pH 5, the nanoparticles are unstable, driving acylated pHD108 to bind strongly to membranes. We hypothesize that passive endocytosis of macromolecular cargo and stable peptide nanoparticles, followed by endosomal acidification-dependent destabilization of the nanoparticles, triggers the nanopore-forming activity of acylated pHD peptides in the endosomal membrane, enabling internalized macromolecules to be delivered to the cytosol.