Mechanisms of lipoplex formation: dependence of the biological properties of transfection complexes on formulation procedures.

Phospholipid-DNA complexes were made of the cationic triester derivative of phosphatidylcholine, EDOPC (1,2-dioleoyl- sn-glycero-3-ethylphosphocholine), by varying conditions of complex formation, in particular, the rate and direction of mixing, as well as by changing the mode of dispersing the lipid (extrusion or vortexing). The biological effects of variations in the formulation procedure were assessed by measuring transfection activity and cell association in cultures of BHK cells. Formulation procedures generally had little effect on cell association, but had marked effects on transfection efficiency. Transfection varied from effectively nil to extremely efficient with what appeared to be modest changes in formulation procedure. Formulation procedures also had significant effects on average sizes and size distributions of lipoplexes as determined by dynamic light scattering. Among the four possibilities of rapid or slow mixing combined with the two possible directions of mixing, slow addition of DNA to lipid gave results that differed significantly from the other three modes. In the case of vortexed lipid, the latter procedure was much less satisfactory than the other three, whereas in the case of extruded lipid, it was the only mode that produced satisfactory transfection. The factors that determine the difference in lipoplex properties can be identified as both geometric and physical. The geometric factor has to do with the symmetries of the participating units. There are three physical factors that are critical: the difference in vesicle stability upon interaction with DNA, the time dependence of interdiffusion of the components relative to that of vesicle rupture, and difference in input concentrations. These factors determine lipoplex size and, as already also shown by others, lipoplex size influences transfection efficiency.

Free energies of urea and of thermal unfolding show that two tandem repeats of spectrin are thermodynamically more stable than a single repeat.

Free energies of both urea and thermal denaturation have been measured for three pairs of one- and two-repeat fragments, cloned in tandem from the cytoskeletal protein, alpha-spectrin, from chicken brain to ascertain whether one- and two-repeat fragments are equally stable. One- and two-repeat fragments of each pair were designed with the same N-terminus, whereas the C-terminus of the two-repeat fragment was 106 residues or the length of one repeat downstream from that of the one-repeat fragment. The averaged free energies of urea and thermal denaturation of the paired fragments, (R16)(00) and (R16R17)(00), (R16)(0+3) and (R16R17)(0+3), and (R16)(+8-4) and (R16R17)(+8-4) [subscripts represent the N- and C-terminal positions with "00" referring to the N- and C-termini defining a repeat according to X-ray crystal structures of two repeat fragments [Grum, V. L., Li, D., MacDonald, R. I., and Mondragón, A. (1999) Cell 98, 523-535] and "+" and "-" referring to positions upstream and downstream therefrom, respectively], increased from 3.7 +/- 0.4 kcal/mol for (R16)(00), 3.7 +/- 0.5 kcal/mol for (R16)(0+3), 4.4 +/- 0.4 kcal/mol for (R16)(+8-4), 6.2 +/- 0.6 kcal/mol for (R16R17)(+8-4), 8.3 +/- 0.4 kcal/mol for (R16R17)(00) to 9.9 +/- 1.0 kcal/mol for (R16R17)(0+3). Thus, the two-repeat fragment of each pair was significantly more thermodynamically stable than the single repeat by both urea and thermal denaturation. Differences in phasing among single repeats did not have the same effect as the same differences in phasing among two-repeat fragments. Addition of nine residues to the C-terminus of (R16R17)(00) yielded a free energy of unfolding of 7.9 +/- 0.8 kcal/mol, whereas addition of seven residues to the C-terminus of (R16)(+8-4) yielded a free energy of unfolding of 5.9 +/- 0.3 kcal/mol.

Factors governing the assembly of cationic phospholipid-DNA complexes.

The interaction of DNA with a novel cationic phospholipid transfection reagent, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC), was investigated by monitoring thermal effects, particle size, vesicle rupture, and lipid mixing. By isothermal titration calorimetry, the heat of interaction between large unilamellar EDOPC vesicles and plasmid DNA was endothermic at both physiological and low ionic strength, although the heat absorbed was slightly larger at the higher ionic strength. The energetic driving force for DNA-EDOPC association is thus an increase in entropy, presumably due to release of counterions and water. The estimated minimum entropy gain per released counterion was 1.4 cal/mole- degrees K (about 0.7 kT), consistent with previous theoretical predictions. All experimental approaches revealed significant differences in the DNA-lipid particle, depending upon whether complexes were formed by the addition of DNA to lipid or vice versa. When EDOPC vesicles were titrated with DNA at physiological ionic strength, particle size increased, vesicles ruptured, and membrane lipids became mixed as the amount of DNA was added up to a 1.6:1 (+:-) charge ratio. This charge ratio also corresponded to the calorimetric end point. In contrast, when lipid was added to DNA, vesicles remained separate and intact until a charge ratio of 1:1 (+:-) was exceeded. Under such conditions, the calorimetric end point was 3:1 (+:-). Thus it is clear that fundamental differences in DNA-cationic lipid complexes exist, depending upon their mode of formation. A model is proposed to explain the major differences between these two situations. Significant effects of ionic strength were observed; these are rationalized in terms of the model. The implications of the analysis are that considerable control can be exerted over the structure of the complex by exploiting vectorial preparation methods and manipulating ionic strength.

Physical and biological properties of cationic triesters of phosphatidylcholine.

The properties of a new class of phospholipids, alkyl phosphocholine triesters, are described. These compounds were prepared from phosphatidylcholines through substitution of the phosphate oxygen by reaction with alkyl trifluoromethylsulfonates. Their unusual behavior is ascribed to their net positive charge and absence of intermolecular hydrogen bonding. The O-ethyl, unsaturated derivatives hydrated to generate large, unilamellar liposomes. The phase transition temperature of the saturated derivatives is very similar to that of the precursor phosphatidylcholine and quite insensitive to ionic strength. The dissociation of single molecules from bilayers is unusually facile, as revealed by the surface activity of aqueous liposome dispersions. Vesicles of cationic phospholipids fused with vesicles of anionic lipids. Liquid crystalline cationic phospholipids such as 1, 2-dioleoyl-sn-glycero-3-ethylphosphocholine triflate formed normal lipid bilayers in aqueous phases that interacted with short, linear DNA and supercoiled plasmid DNA to form a sandwich-structured complex in which bilayers were separated by strands of DNA. DNA in a 1:1 (mol) complex with cationic lipid was shielded from the aqueous phase, but was released by neutralizing the cationic charge with anionic lipid. DNA-lipid complexes transfected DNA into cells very effectively. Transfection efficiency depended upon the form of the lipid dispersion used to generate DNA-lipid complexes; in the case of the O-ethyl derivative described here, large vesicle preparations in the liquid crystalline phase were most effective.