Cationic cell-penetrating peptides induce ceramide formation via acid sphingomyelinase: implications for uptake

J Control Release. 2010 Oct 15;147(2):171-9. doi: 10.1016/j.jconrel.2010.06.030. Epub 2010 Jul 8.


Cationic cell-penetrating peptides (CPP) are receiving increasing attention as molecular transporters of membrane-impermeable molecules. Import of cationic CPP occurs both via endocytosis and - at higher peptide concentrations - in an endocytosis-independent manner via localized regions of the plasma membrane. At present, this endocytosis-independent import of cationic CPP is not well understood, but has been shown to be sensitive to various pharmacological inhibitors, suggesting a role of an unidentified enzymatic activity. Here, we demonstrate that the direct translocation of cationic CPP depends on a CPP-induced translocation of acid sphingomyelinase (ASMase) to the outer leaflet of the plasma membrane and ceramide formation. The involvement of ASMase in uptake was confirmed by a pharmacological inhibition of ASMase by imipramine and a subsequent rescue of uptake through external addition of sphingomyelinase, and by using ASMase-deficient cells. We also found that the threshold for direct CPP translocation can be lowered through addition of sphingomyelinase and that sphingomyelinase enhances the translocation of R9 coupled to low-molecular weight cargos, but not high-molecular weight cargos. In conclusion, we show that a previously poorly understood mechanism of cationic CPP import depends on the ASMase-dependent formation of ceramide on the outer leaflet of the plasma membrane. To our knowledge, this is the first illustration that a class of delivery vectors operates through the induction of an enzymatic activity that changes the lipid composition of the plasma membrane.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cations
  • Cell Culture Techniques
  • Cell Membrane / enzymology*
  • Cell Membrane / metabolism
  • Cell Membrane Permeability / drug effects
  • Cell-Penetrating Peptides / chemistry
  • Cell-Penetrating Peptides / pharmacokinetics
  • Cell-Penetrating Peptides / pharmacology*
  • Ceramides / metabolism*
  • Drug Carriers / chemistry
  • Drug Carriers / pharmacokinetics
  • Drug Carriers / pharmacology*
  • Enzyme Inhibitors / pharmacology
  • Fibroblasts / drug effects
  • Fibroblasts / enzymology
  • Fibroblasts / metabolism
  • HeLa Cells
  • Humans
  • Mice
  • Mice, Knockout
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • Protein Transport
  • Sphingomyelin Phosphodiesterase / antagonists & inhibitors
  • Sphingomyelin Phosphodiesterase / genetics
  • Sphingomyelin Phosphodiesterase / metabolism*


  • Cations
  • Cell-Penetrating Peptides
  • Ceramides
  • Drug Carriers
  • Enzyme Inhibitors
  • Sphingomyelin Phosphodiesterase