Programmable fusogenic vesicles for intracellular delivery of antisense oligodeoxynucleotides: enhanced cellular uptake and biological effects

Programmable fusogenic vesicles (PFV) are liposomes composed of non-bilayer lipid components stabilized by the inclusion of an exchangeable poly(ethyl ene glycol) (PEG)-lipid conjugate. Vesicle destabilization by loss of the P EG-lipid results in recovery of the inherent fusogenic character. As a resu lt, PFV can be designed to display a long circulation lifetime after i.v. a dministration, high accumulation at disease sites and full bioavailability of an encapsulated compound. In the present study, we investigated the pote ntial application of PFV as carriers for intracellular delivery of antisens e oligodeoxynucleotides (ODN). Antisense phosphorothioate ODN were encapsul ated into PFV containing dioleoylphosphatidylethanolamine, cholesterol, dio leyldimethylammonium chloride and PEG-ceramides with different carbon chain length (C-8, C-14 and C-20). In vitro fluorescent microscopy and flow cyto metry analysis demonstrated that PFV containing PEG-ceramide C-14 provided enhanced intracellular delivery of FITC-labelled antisense ODN compared to PFV displaying faster or slower rates of destabilization (containing PEG-ce ramide C-8 or C-20, respectively). Therapeutic efficacy of PFV-encapsulated antisense ODN against two proto-oncogenes, c-myc and bcl-2, was examined i n various cell lines. At antisense concentrations of 0.5 muM, no significan t downregulation of c-myc mRNA levels was observed in HEK293, B16 and MCA20 7 cells. However, treatment of 518A2 melanoma cells with PFV-encapsulated a ntisense targeting bcl-2 at concentrations of 0.5 muM and 1.0 muM resulted in reduced bcl-2 mRNA level by about 20% and 25% after 48 h incubation. Fre e antisense ODN did not affect bcl-2 mRNA expression at the concentrations used in this study and encapsulated control antisense (reverse polarity) le d to a non-specific increase in mRNA levels. Our results suggest that PFV c arriers displaying appropriate rates of destabilization have the potential to act as intracellular delivery vehicles and may improve the bioavailabili ty and potency of antisense oligonucleotides. (C) 2001 Elsevier Science B.V . All rights reserved.