The use of fluorescence resonance energy transfer to monitor dynamic changes of lipid-DNA interactions during lipoplex formation.
Fluorescence resonance energy transfer (FRET) was used to monitor interactions between Cy3-labeled plasmid DNA and NBD-labeled cationic liposomes. FRET data show that binding of cationic liposomes to DNA occurs immediately upon mixing (within 1 min), but FRET efficiencies do not stabilize for 1-5 h. The time allowed for complex formation has effects on in vitro luciferase transfection efficiencies of DOPE-based lipoplexes; i.e., lipoplexes prepared with a 1-h incubation have much higher transfection efficiencies than samples with 1-min or 5-h incubations. The molar charge ratio of DOTAP to negatively charged phosphates in the DNA (DOTAP+/DNA-) also affected the interaction between liposomes and plasmid DNA, and interactions stabilized more rapidly at higher charge ratios. Lipoplexes formulated with DOPE were more resistant to high ionic strength than complexes formulated with cholesterol. Taken together, our data demonstrate that lipid-DNA interactions and in vitro transfection efficiencies are strongly affected by the time allowed for complex formation. This effect is especially evident in DOPE-based lipoplexes, and suggests that the time allowed for lipoplex formation is a parameter that should be carefully controlled in future studies.
Zhang
Ye
Y
Department of Pharmaceutical Sciences, School of Pharmacy, C238, University of Colorado Health Sciences Center, 4200 E. Ninth Avenue, Denver, CO 80262, USA. Ye.Zhang@UCHSC.edu
Garzon-Rodriguez
William
W
Manning
Mark C
MC
Anchordoquy
Thomas J
TJ
D002232:Carbocyanines; D004247:DNA / Q000737:chemistry*; D005229:Fatty Acids, Monounsaturated; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D008055:Lipids / Q000737:chemistry*; D008081:Liposomes / Q000138:chemical synthesis*; D009994:Osmolar Concentration; D010316:Particle Size; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; D012965:Sodium Chloride / Q000737:chemistry; D014162:Transfection / Q000379:methods
D002232:Carbocyanines; D005229:Fatty Acids, Monounsaturated; D005456:Fluorescent Dyes; D008055:Lipids; D008081:Liposomes; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; C089187:cyanine dye 3; D012965:Sodium Chloride; C042077:N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine; D004247:DNA; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane