A multi-step lipid mixing assay to model structural changes in cationic lipoplexes used for in vitro transfection.
Formation of liposome/polynucleotide complexes (lipoplexes) involves electrostatic interactions, which induce changes in liposome structure. The ability of these complexes to transfer DNA into cells is dependent on the physicochemical attributes of the complexes, therefore characterization of binding-induced changes in liposomes is critical for the development of lipid-based DNA delivery systems. To clarify the apparent lack of correlation between membrane fusion and in vitro transfection previously observed, we performed a multi-step lipid mixing assay to model the sequential steps involved in transfection. The roles of anion charge density, charge ratio and presence of salt on lipid mixing and liposome aggregation were investigated. The resonance-energy transfer method was used to monitor lipid mixing as cationic liposomes (DODAC/DOPE and DODAC/DOPC; 1:1 mole ratio) were combined with plasmid, oligonucleotides or Na(2)HPO(4). Cryo-transmission electron microscopy was performed to assess morphology. As plasmid or oligonucleotide concentration increased, lipid mixing and aggregation increased, but with Na(2)HPO(4) only aggregation occurred. NaCl (150 mM) reduced the extent of lipid mixing. Transfection studies suggest that the presence of salt during complexation had minimal effects on in vitro transfection. These data give new information about the effects of polynucleotide binding to cationic liposomes, illustrating the complicated nature of anion induced changes in liposome morphology and membrane behavior.
Wasan
E K
EK
Advanced Therapeutics-Medical Oncology, BC Cancer Agency, 600 West 10th Ave., Vancouver, BC, Canada.
Harvie
P
P
Edwards
K
K
Karlsson
G
G
Bally
M B
MB
D009327:4-Chloro-7-nitrobenzofurazan; D000818:Animals; D002412:Cations; D020285:Cryoelectron Microscopy; D005456:Fluorescent Dyes; D008081:Liposomes / Q000737:chemistry* / Q000648:ultrastructure; D051379:Mice; D008958:Models, Molecular; D015394:Molecular Structure; D010316:Particle Size; D010957:Plasmids / Q000737:chemistry*; D011119:Polynucleotides / Q000737:chemistry*; D012965:Sodium Chloride; D013329:Structure-Activity Relationship; D014162:Transfection; D014407:Tumor Cells, Cultured
D002412:Cations; D005456:Fluorescent Dyes; D008081:Liposomes; D011119:Polynucleotides; D012965:Sodium Chloride; D009327:4-Chloro-7-nitrobenzofurazan