title: Enhanced delivery of cell-penetrating peptide-peptide nucleic acid conjugates by endosomal disruption.
abstract: Improvement of cellular uptake and cellular localization is still one of the main obstacles to the development of antisense-antigene therapeutics, including peptide nucleic acid (PNA). Cell-penetrating peptides (CPPs) such as Tat peptide and polyarginine have been widely used to improve the cellular uptake of PNA and other antisense agents. Cellular uptake of most CPP conjugates occurs mainly through endocytotic pathways, and most CPP conjugate is retained in the endosomal compartments of the cell. Several methods to induce endosome disruption have been shown to improve the bioavailability of CPP conjugates to the cytosol and/or nucleus by facilitating escape from the endosomal compartments. Here we describe protocols for the delivery of CPP-PNA conjugates to adherent cultured cells using photodynamic treatment (photochemical internalization), Ca2+ treatment or chloroquine treatment to potentiate the antisense effects of CPP-PNA conjugates through increased release of CPP conjugates into the cytoplasm. This protocol, consisting of CPP-mediated delivery assisted by an endosome-disruption agent, allows the delivery of the CPP-PNA conjugates to the nucleus and/or cytosol of cultured cells. The endosome-disruption treatment improves the nuclear antisense effects of CPP-PNA conjugates by up to two orders of magnitude using 24-hour delivery.
authors.affiliation: Department of Medical Biochemistry and Genetics, The Panum Institute, University of Copenhagen, Blegdamsvej 3c, 2200 Copenhagen, Denmark. shiraishi@imbg.ku.dk
authors.forename: Takehiko
authors.initials: T
authors.lastname: Shiraishi
authors.forename: Peter E
authors.initials: PE
authors.lastname: Nielsen
mesh_terms: D002118:Calcium; D002467:Cell Nucleus / Q000378:metabolism; D002738:Chloroquine; D011992:Endosomes / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D006367:HeLa Cells; D006801:Humans; D020135:Peptide Nucleic Acids / Q000737:chemistry* / Q000378:metabolism*; D010777:Photochemistry
chemical_list: D020135:Peptide Nucleic Acids; D002738:Chloroquine; D002118:Calcium