This study seeks correlations between the molecular structures of cationic and 
neutral lipids, the lipid phase behavior of the mixed-lipid lipoplexes they form 
with plasmid DNA, and the transfection efficacy of the lipoplexes. Synthetic 
cationic pyridinium lipids were co-formulated (1:1) with the cationic lipid 
1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC), and these lipids were 
co-formulated (3:2) with the neutral lipids 
1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) or cholesterol. All 
lipoplex formulations exhibited plasmid DNA binding and a level of protection 
from DNase I degradation. Composition-dependent transfection (beta-galactosidase 
and GFP) and cytotoxicity was observed in Chinese hamster ovarian-K1 cells. The 
most active formulations containing the pyridinium lipids were less cytotoxic 
but of comparable activity to a Lipofectamine 2000™ control. Molecular structure 
parameters and partition coefficients were calculated for all lipids using 
fragment additive methods. The derived shape parameter values correctly 
correlated with observed hexagonal lipid phase behavior of lipoplexes as derived 
from small-angle X-ray scattering experiments. A transfection index applicable 
to hexagonal phase lipoplexes derived from calculated parameters of the lipid 
mixture (partition coefficient, shape parameter, lipoplex packing) produced a 
direct correlation with transfection efficiency.