Delivery of steric block morpholino oligomers by (R-X-R)(4) peptides: structure–activity studies

Abes, Rachida; Moulton, Hong M.; Clair, Philippe; Yang, Sung-Tae; Abes, Said; Melikov, Kamran; Prevot, Paul; Youngblood, Derek S.; Iversen, Patrick L.; Chernomordik, Leonid V.; Lebleu, Bernard

Large unilamellar vesicles (LUV) were prepared as described previously (16) . In short, lipids dissolved in benzene/methanol (95:5) were freeze-dried overnight and the resulting dry lipid powder was hydrated in a buffer containing the ANTS fluorescent dye 8-aminonaphthalene-1,3,6-trisulfonic acid, disodium salt (Invitrogen, Carlsbad, CA, USA) together with a DPX quencher p-xylene-bispyridinium bromide (Invitrogen, Carlsbad, CA, USA) at a final lipid concentration of 10 mM. The suspension was vigorously agitated with a Vortex, freeze-thawed 10 times and then extruded 10 times through two stacked 100 nm polycarbonate filters (Nucleopore, Whatman). Free dye and quencher were then removed by gel filtration on a PD-10 desalting column (Amersham Biosciences, Piscataway, NJ, USA). To mimic the lipid composition of late endosomes we used the following lipid mixture: dioleoylphosphatidylcholine (DOPC)/dioleoyl-phosphatidylethanolamine (DOPE)/phosphatidylinositol from soybean (PI)/bis(monooleoylglycero) phosphate (LBPA) (5:2:1:2) (23). All lipids were purchased from Avanti Polar Lipids Inc., Alabaster, AL.