title: Multivalent presentation of the cell-penetrating peptide nona-arginine on a linear scaffold strongly increases its membrane-perturbing capacity.
abstract: Arginine-rich cell-penetrating peptides (CPP) are widely employed as delivery vehicles for a large variety of macromolecular cargos. As a mechanism-of-action for induction of uptake cross-linking of heparan sulfates and interaction with lipid head groups have been proposed. Here, we employed a multivalent display of the CPP nona-arginine (R9) on a linear dextran scaffold to assess the impact of heparan sulfate and lipid interactions on uptake and membrane perturbation. Increased avidity through multivalency should potentiate molecular phenomena that may only play a minor role if only individual peptides are used. To this point, the impact of multivalency has only been explored for dendrimers, CPP-decorated proteins and nanoparticles. We reasoned that multivalency on a linear scaffold would more faithfully mimic the arrangement of peptides at the membrane at high local peptide concentrations. On average, five R9 were coupled to a linear dextran backbone. The conjugate displayed a direct cytoplasmic uptake similar to free R9 at concentrations higher than 10μM. However, this uptake was accompanied by an increased membrane disturbance and cellular toxicity that was independent of the presence of heparan sulfates. In contrast, for erythrocytes, the multivalent conjugate induced aggregation, however, showed only limited membrane perturbation. Overall, the results demonstrate that multivalency of R9 on a linear scaffold strongly increases the capacity to interact with the plasma membrane. However, the induction of membrane perturbation is a function of the cellular response to peptide binding.
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Geert Grooteplein 28, 6525 GA Nijmegen, the Netherlands.
authors.forename: Alokta
authors.initials: A
authors.lastname: Chakrabarti
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Geert Grooteplein 28, 6525 GA Nijmegen, the Netherlands.
authors.forename: J Joris
authors.initials: JJ
authors.lastname: Witsenburg
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Geert Grooteplein 28, 6525 GA Nijmegen, the Netherlands.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Sinzinger
authors.affiliation: Leibniz Institute for Molecular Pharmacology (FMP), Robert-Rössle-Str. 10, 13125 Berlin, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Richter
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Geert Grooteplein 28, 6525 GA Nijmegen, the Netherlands.
authors.forename: Rike
authors.initials: R
authors.lastname: Wallbrecher
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Geert Grooteplein 28, 6525 GA Nijmegen, the Netherlands.
authors.forename: Judith C
authors.initials: JC
authors.lastname: Cluitmans
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Geert Grooteplein 28, 6525 GA Nijmegen, the Netherlands.
authors.forename: Wouter P R
authors.initials: WP
authors.lastname: Verdurmen
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Geert Grooteplein 28, 6525 GA Nijmegen, the Netherlands.
authors.forename: Sabine
authors.initials: S
authors.lastname: Tanis
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Geert Grooteplein 28, 6525 GA Nijmegen, the Netherlands.
authors.forename: Merel J W
authors.initials: MJ
authors.lastname: Adjobo-Hermans
authors.affiliation: Leibniz Institute for Molecular Pharmacology (FMP), Robert-Rössle-Str. 10, 13125 Berlin, Germany; Medicinal Chemistry, Free University Berlin, Königin-Luise-Str. 2+4, 14195 Berlin, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Rademann
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Geert Grooteplein 28, 6525 GA Nijmegen, the Netherlands. Electronic address: r.brock@ncmls.ru.nl.
authors.forename: Roland
authors.initials: R
authors.lastname: Brock
keywords: Cell-penetrating peptide; Drug delivery; Multivalency; Nona-arginine; Protein–protein interactions; Sphingomyelinase