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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 cationic lipid-nucleic acid-cell interactions. Fluorescent confocal microscopy of cells treated with rhodamine-labeled cationic liposome/fluorescein-labeled ODN (...

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Published in:Proceedings of the National Academy of Sciences - PNAS 1996-10, Vol.93 (21), p.11493-11498
Main Authors: Zelphati, Olivier, Szoka, Francis C.
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Szoka, Francis C.
description We propose a mechanism for oligonucleotide (ODN) release from cationic lipid complexes in cells that accounts for various observations on cationic lipid-nucleic acid-cell interactions. Fluorescent confocal microscopy of cells treated with rhodamine-labeled cationic liposome/fluorescein-labeled ODN (F-ODN) complexes show the F-ODN separates from the lipid after internalization and enters the nucleus leaving the fluorescent lipid in cytoplasmic structures. ODN displacement from the complex was studied by fluorescent resonance energy transfer. Anionic liposome compositions (e.g., phosphatidylserine) that mimic the cytoplasmic facing monolayer of the cell membrane released ODN from the complex at about a 1:1 (-/+) charge ratio. Release was independent of ionic strength and pH. Physical separation of the F-ODN from monovalent and multivalent cationic lipids was confirmed by gel electrophoresis. Fluid but not solid phase anionic liposomes are required, whereas the physical state of the cationic lipids does not effect the release. Water soluble molecules with a high negative linear charge density, dextran sulfate, or heparin also release ODN. However, ATP, spermidine, spermine, tRNA, DNA, polyglutamic acid, polylysine, bovine serum albumin, or histone 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 cytoplasmic facing monolayer. Anionic lipids laterally diffuse into the complex and form a charged neutralized ion-pair with the cationic lipids. This leads to displacement of the ODN from the cationic lipid and its release into the cytoplasm.
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Fluorescent confocal microscopy of cells treated with rhodamine-labeled cationic liposome/fluorescein-labeled ODN (F-ODN) complexes show the F-ODN separates from the lipid after internalization and enters the nucleus leaving the fluorescent lipid in cytoplasmic structures. ODN displacement from the complex was studied by fluorescent resonance energy transfer. Anionic liposome compositions (e.g., phosphatidylserine) that mimic the cytoplasmic facing monolayer of the cell membrane released ODN from the complex at about a 1:1 (-/+) charge ratio. Release was independent of ionic strength and pH. Physical separation of the F-ODN from monovalent and multivalent cationic lipids was confirmed by gel electrophoresis. Fluid but not solid phase anionic liposomes are required, whereas the physical state of the cationic lipids does not effect the release. Water soluble molecules with a high negative linear charge density, dextran sulfate, or heparin also release ODN. However, ATP, spermidine, spermine, tRNA, DNA, polyglutamic acid, polylysine, bovine serum albumin, or histone 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 cytoplasmic facing monolayer. Anionic lipids laterally diffuse into the complex and form a charged neutralized ion-pair with the cationic lipids. This leads to displacement of the ODN from the cationic lipid and its release into the cytoplasm.</abstract><cop>United States</cop><pub>National Academy of Sciences of the United States of America</pub><pmid>8876163</pmid><doi>10.1073/pnas.93.21.11493</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record>
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1091-6490
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source PubMed Central Free; JSTOR Archival Journals and Primary Sources Collection
subjects Base Sequence
Biochemistry
Cell Membrane
Cell membranes
Dextrans
DNA
Endocytosis
Energy Transfer
Fluorescence
Gels
Heparin
Hydrogen-Ion Concentration
Lipids
Liposomes
Microscopy, Confocal
Models, Biological
Molecules
Nucleic acids
Oligodeoxyribonucleotides - chemistry
Osmolar Concentration
RNA, Transfer
Serum Albumin, Bovine
Spectrometry, Fluorescence
Spermidine
Spermine
Structure-Activity Relationship
Sulfates
Thionucleotides
title Mechanism of Oligonucleotide Release from Cationic Liposomes
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