title: Improved cellular activity of antisense peptide nucleic acids by conjugation to a cationic peptide-lipid (CatLip) domain.
abstract: Conjugation to cationic cell penetrating peptides (such as Tat, Penetratin, or oligo arginines) efficiently improves the cellular uptake of large hydrophilic molecules such as oligonucleotides and peptide nucleic acids, but the cellular uptake is predominantly via an unproductive endosomal pathway and therefore mechanisms that promote endosomal escape (or avoid the endosomal route) are required for improving bioavailability. A variety of auxiliary agents (chloroquine, calcium ions, or lipophilic photosensitizers) has this effect, but improved, unaided delivery would be highly advantageous in particular for future in vivo applications. We find that simply conjugating a lipid domain (fatty acid) to the cationic peptide (a CatLip conjugate) increases the biological effect of the corresponding PNA (CatLip) conjugates in a luciferase cellular antisense assay up to 2 orders of magnitude. The effect increases with increasing length of the fatty acid (C8-C16) but in parallel also results in increased cellular toxicity, with decanoic acid being optimal. Furthermore, the relative enhancement is significantly higher for Tat peptide compared to oligoarginine. Confocal microscopy and chloroquine enhancement indicates that the lipophilic domain increases the endosomal uptake as well as promoting significantly endosomal escape. These results provide a novel route for improving the (cellular) bioavailability of larger hydrophilic molecules.
authors.affiliation: Department of Cellular and Molecular Medicine, The Panum Institute, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3c, DK-2200 Copenhagen N, Denmark.
authors.forename: Uffe
authors.initials: U
authors.lastname: Koppelhus
authors.forename: Takehiko
authors.initials: T
authors.lastname: Shiraishi
authors.forename: Vladimir
authors.initials: V
authors.lastname: Zachar
authors.forename: Stanislava
authors.initials: S
authors.lastname: Pankratova
authors.forename: Peter E
authors.initials: PE
authors.lastname: Nielsen
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism; D002412:Cations / Q000737:chemistry; D002477:Cells / Q000166:cytology / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism; D015696:Gene Products, tat / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D020135:Peptide Nucleic Acids / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary
chemical_list: D002412:Cations; D005227:Fatty Acids; D015696:Gene Products, tat; D008055:Lipids; D016376:Oligonucleotides, Antisense; D020135:Peptide Nucleic Acids; D010455:Peptides; D001120:Arginine; D008156:Luciferases