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 electr ostatic interactions, which induce changes in liposome structure. The abili ty of these complexes to transfer DNA into cells is dependent on the physic ochemical attributes of the complexes, therefore characterization of bindin g-induced changes in liposomes is critical for the development of lipid-bas ed DNA delivery systems. To clarify the apparent lack of correlation betwee n membrane fusion and in vitro transfection previously observed, we perform ed a multi-step lipid mixing assay to model the sequential steps involved i n transfection. The roles of anion charge density, charge ratio and presenc e of salt on lipid mixing and liposome aggregation were investigated. The r esonance-energy transfer method was used to monitor lipid mixing as cationi c liposomes (DODAC/DOPE and DODAC/DOPC; 1:1 mole ratio) were combined with plasmid, oligonucleotides or Na2HPO4. Cryo-transmission electron microscopy was performed to assess morphology. As plasmid or oligonucleotide concentr ation increased, lipid mixing and aggregation increased, but with Na2HPO4 o nly 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 informat ion about the effects of polynucleotide binding to cationic liposomes, illu strating the complicated nature of anion induced changes in liposome morpho logy and membrane behavior. (C) 1999 Elsevier Science B.V. All rights reser ved.