title: Evaluation of cell-penetrating peptides (CPPs) as vehicles for intracellular delivery of antisense peptide nucleic acid (PNA).
abstract: Cell-penetrating peptides (CPPs) are characterized by their ability to be internalized in mammalian cells. To investigate the relative potency of CPPs as carriers of medicinally relevant cargo, a positive read-out assay based on the ability of a peptide nucleic acid (PNA) oligomer to promote correct expression of a recombinant luciferase gene was employed. Seven different CPPs were included in the study: Transportan, oligo-arginine (R7-9), pTat, Penetratin, KFF, SynB3, and NLS. The CPP-PNA conjugates were synthesized by different conjugation chemistries: continuous synthesis, maleimide coupling, and ester or disulfide linkage. Under serum-free conditions PNA-SS-Transportan-amide (ortho)-PNA was found to be the most potent conjugate, resulting in maximum luciferase signal at a concentration of 1-2 microM. (D-Arg)9-PNA showed optimal efficacy at 5 microM but gave rise to only one-third of the luciferase signal obtained with the Transportan conjugate. The pTat- and KFF-PNA conjugates showed significantly lower efficacy. The penetratin-, SynB3-. and NLS-PNA conjugates showed only minimal or no activity. Serum was found to have a drastic negative impact on CPP-driven cellular uptake. PNA-SS-Transportan-acid (ortho) and (D-Arg)9-PNA were least sensitive to the presence of serum. Both the chemical nature and, in the case of Transportan, the position of the peptide PNA coupling were found to have a major impact on the transport capacity of the peptides. However, no simple relationship between linker type and antisense activity of the conjugates could be deduced from the data.
authors.affiliation: Center for Biomolecular Recognition, Department of Medical Biochemistry and Genetics, Biochemistry Laboratory B, The Panum Institute, University of Copenhagen, Blegdamsvej 3c, DK-2200 Copenhagen N, Denmark.
authors.forename: Nadia
authors.initials: N
authors.lastname: Bendifallah
authors.forename: Frank Winther
authors.initials: FW
authors.lastname: Rasmussen
authors.forename: Vladimir
authors.initials: V
authors.lastname: Zachar
authors.forename: Peter
authors.initials: P
authors.lastname: Ebbesen
authors.forename: Peter E
authors.initials: PE
authors.lastname: Nielsen
authors.forename: Uffe
authors.initials: U
authors.lastname: Koppelhus
mesh_terms: D001120:Arginine / Q000737:chemistry; D002463:Cell Membrane Permeability / Q000187:drug effects; D003432:Cross-Linking Reagents / Q000737:chemistry; D016895:Culture Media, Serum-Free; D004952:Esters / Q000737:chemistry; D019004:Galanin / Q000633:toxicity; D006367:HeLa Cells; D006801:Humans; D015394:Molecular Structure; D016376:Oligonucleotides, Antisense / Q000737:chemistry* / Q000494:pharmacology*; D020135:Peptide Nucleic Acids / Q000737:chemistry* / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000633:toxicity; D044967:Serum; D014862:Wasp Venoms / Q000633:toxicity
chemical_list: D003432:Cross-Linking Reagents; D016895:Culture Media, Serum-Free; D004952:Esters; D016376:Oligonucleotides, Antisense; D020135:Peptide Nucleic Acids; D011993:Recombinant Fusion Proteins; D014862:Wasp Venoms; C110068:transportan; D019004:Galanin; D001120:Arginine