Characterization of DNA–lipid complexes commonly used for gene delivery

Abstract

Cationic liposomes are used to deliver genes into cells. Here we describe some poorly understood basic features of DNA–lipid complexes (lipoplexes), especially the electrostatics, stability and DNA structure of lipoplexes, and their effects on transfection (lipofection). Use of the lipophilic, pH-sensitive fluorophore 4-heptadecyl-7-hydroxycoumarin, in combination with Gouy–Chapman calculations, showed that cationic liposomes had a large positive surface potential (180–240 mV) and a high pH (10–11.5) at the location of the probe on the liposomal surface in 20 mM Hepes buffer (pH 7.4). Other electrostatic characteristics were also found, such as the existence of protonable groups of cationic or helper lipids or salt bridges between those. Addition of DNA resulted in neutralization of cationic lipids, which was lower than expected and depending on the type of lipid and the DNA/cationic lipid ratio. The liposomes containing DOTAP ( N-(1-(2,3-dioleoyloxy)propyl)- N,N,N-trimethylammonium chloride) were unstable upon dilution, probably due to the high critical micellar concentration of DOTAP, 7×10 −5 M. Large instablility expressed as continuous size increase was demonstrated by the time-dependent static changes in light-scattering monitored following the mixing of cationic liposomes and DNA at DNA/cationic lipid molar ratios between 0.2 and 0.8. Addition of cationic liposomes composed of 100% DOTAP or DOTAP/DOPE (1/1) liposomes, induced instantaneous transition of the plasmid DNA from the B- toward a partial C-type conformation as shown by circular dichroism (CD) spectroscopy and at certain conditions Ψ −-DNA could be found as well. The Ψ −-DNA is characterized by inter-helical interaction between parallel helices. The highest lipofection was obtained under conditions of lipoplex instability, and when the DNA was partially dehydrated and had a partial Ψ −- structure.