MECHANISM OF OLIGONUCLEOTIDE RELEASE FROM CATIONIC LIPOSOMES

We propose a mechanism for oligonucleotide (ODN) release from cationic lipid complexes in cells that accounts for various observations on ca tionic lipid-nucleic acid-cell interactions. Fluorescent confocal micr oscopy of cells treated with rhodamine-labeled cationic liposome/fluor escein-labeled ODN (F-ODN) complexes show the F-ODN separates from the lipid after internalization and enters the nucleus leaving the fluore scent lipid in cytoplasmic structures. ODN displacement from the compl ex was studied by fluorescent resonance energy transfer. Anionic lipos ome Compositions (e.g., phosphatidylserine) that mimic the cytoplasmic facing monolayer of the cell membrane released ODN from the complex a t about a 1:1 (-/+) charge ratio. Release was independent of ionic str ength and pH. Physical separation of the F-ODN from monovalent and mul tivalent cationic lipids was confirmed by gel electrophoresis. Fluid b ut not solid phase anionic liposomes are required, whereas the physica l state of the cationic lipids does not effect the release. Water solu ble molecules with a high negative linear charge density, dextran sulf ate, or heparin also release ODN. However, ATP, spermidine, spermine, tRNA, DNA, polyglutamic acid, polylysine, bovine serum albumin, or his tone did not release ODN, even at 100-fold charge excess (-/+). Based upon these results, we propose that the complex, after internalization by endocytosis, induces flip-flop of anionic lipids from the cytoplas mic facing monolayer. Anionic lipids laterally diffuse into the comple x and form a charged neutralized ion-pair with the cationic lipids. Th is leads to displacement of the ODN from the cationic lipid and its re lease into the cytoplasm.