Novel N,N ′‐diacyl‐1,3‐diaminopropyl‐2‐carbamoyl bivalent cationic lipids for gene delivery – synthesis, in vitro transfection activity, and physicochemical characterization

Novel N,N′‐diacyl‐1,3‐diaminopropyl‐2‐carbamoyl bivalent cationic lipids were synthesized and their physicochemical properties in lamellar assemblies with and without plasmid DNA were evaluated to elucidate the structural requirements of these double‐chained pH‐sensitive surfactants for potent non‐viral gene delivery and expression. The highest in vitro transfection efficacies were induced at +/− 4 : 1 by the dimyristoyl, dipalmitoyl and dioleoyl derivatives 1,3lb2, 1,3lb3 and 1,3lb5, respectively, without inclusion of helper lipids. Transfection activities were reduced in the presence of either 1,2‐dioleoyl‐sn‐glycero‐3‐phosphoethanolamine alone or in combination with cholesterol for all derivatives except 1,3lb5, which maintained reporter gene expression levels at +/− 4 : 1 and yielded increased lipofection activity at a lower charge ratio of +/− 2 : 1. Ethidium bromide displacement indicated efficient plasmid DNA binding and compaction by the transfection‐competent analogs. Dynamic light‐scattering and electrophoretic mobility studies revealed lipoplexes of the active lipids with large particle sizes (mean diameter ≥ 500 nm) and zeta potentials with positive values (low ionic strength) or below neutrality (high ionic strength). Langmuir film balance studies showed high in‐plane elasticity of these derivatives in isolation. In agreement with the monolayer experiments, fluorescence polarization studies verified the fluid nature of the highly transfection‐efficient amphiphiles, with gel‐to‐liquid crystalline phase transitions below physiological temperature. The active compounds also interacted with endosome‐mimicking vesicles to a greater extent than the poorly active derivative 1,3lb4, as revealed by fluorescence resonance energy transfer experiments. Taken together, the results suggest that well‐hydrated and highly elastic cationic lipids with increased acyl chain fluidity and minimal cytotoxicity elicit high transfection activity.