Reducible cationic lipids for gene transfer

One of the main challenges of gene therapy remains the increase of gene del ivery into eukaryotic cells. We tested whether intracellular DNA release, a n essential step for gene transfer, could be facilitated by using reducible cationic DNA-delivery vectors. For this purpose, plasmid DNA was complexed with cationic lipids bearing a disulphide bond, This reduction-sensitive l inker is expected to be reduced and cleaved in the reducing milieu of the c ytoplasm, thus potentially improving DNA release and consequently transfect ion, The DNA-disulphide-lipid complexation was monitored by ethidium bromid e exclusion, and the size of complexes was determined by dynamic light scat tering, It was found that the reduction kinetics of disulphide groups in DN A-lipid complexes depended on the position of the disulphide linker within the lipid molecule, Furthermore, the internal structure of DNA-lipid partic les was examined by small-angle X-ray scattering before and after lipid red uction. DNA release from lipid complexes was observed after the reduction o f disulphide bonds of several lipids. Cell-transfection experiments suggest ed that complexes formed with selected reducible lipids resulted in up to 1 000-fold higher reporter-gene activity, when compared with their analogues without disulphide bonds. In conclusion, reduction-sensitive groups introdu ced into cationic lipid backbones potentially allow enhanced DNA release fr om DNA-lipid complexes after intracellular reduction and represent a tool f or improved vectorization.