Selective Disruption of Early/Recycling Endosomes: Release of Disulfide-Linked Cargo Mediated by a N-Alkyl-3β-Cholesterylamine-Capped Peptide

Abstract

The use of endocytic uptake pathways to deliver poorly permeable molecules into mammalian cells is often plagued by entrapment and degradation of material in late endosomes and lysosomes. As a strategy to prevent the exposure of cargo to these highly hydrolytic membrane-sealed compartments, we synthesized derivatives of the membrane anchor N-alkyl-3β-cholesterylamine that selectively target linked compounds to less hydrolytic early/recycling endosomes. By targeting a pH-dependent membrane-lytic dodecapeptide and a disulfide-linked fluorophore to these compartments in Chinese hamster ovary cells or Jurkat lymphocytes, membranes of early/recycling endosomes were selectively disrupted, resulting in cleavage of the disulfide and escape of the fluorophore into the cytosol and nucleus with low toxicity. The ability of appropriately designed N-alkyl-3β-cholesterylamines to deliver cargo into and release disulfide-linked cargo from relatively nonhydrolytic early/recycling endosomes may be useful for the delivery of a variety of sensitive molecules into living mammalian cells.Receptor-mediated endocytosis (RME) is a major mechanism of uptake of impermeant molecules by mammalian cells. 1 In this process, extracellular ligands bind cell surface receptors that cluster in dynamic regions of cellular plasma membranes. By actively pinching off to form intracellular vesicles, these membrane regions are internalized, encapsulating ligand-receptor complexes in the cytoplasm. These vesicles fuse and form early (primary/ sorting) endosomes that are acidified (pH ∼ 6) by the activation of proton pumps, conditions that generally promote the dissociation of receptors from bound ligands. Free receptors often cycle back to the cell surface, generally via subsequent trafficking through related recycling endosomes (also termed the endocytic recycling compartment). 2 In contrast, free ligands are typically directed to more acidic late endosomes and lysosomes (pH ∼ 5), where hydrolases and other enzymes promote their degradation. Some viruses and other intracellular pathogens exploit RME to enter cells, but these organisms avoid degradation in lysosomes by expressing pH-dependent fusogenic proteins that disrupt endosomal membranes. 3 To escape entrapment within these membranes and gain access to the cytosol, Semliki Forest virus disrupts early endosomes whereas influenza virus disrupts late endosomes during the course of infection. 3Correspondence to: Blake R. Peterson. We report here the synthesis of novel compounds designed to enable membrane-bound disulfide-linked cargo to selectively escape from early/recycling endosomes of living mammalian cells. Because these endosomes are less acidic and less hydrolytically active than late endosomes/lysosomes, this approach may be advantageous when compared to delivery methods that penetrate deeper into the endosomal system. To selectively deliver compounds into early/recycling endosomes, we synthesized four derivatives of the dynamic membrane anchor N-alkyl-3β-cholesterylamine 4,5 (1-4). Tw...

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