Design, Syntheses and In Vitro Gene Delivery Efficacies of Novel Mono-, Di- and Trilysinated Cationic Lipids:  A Structure−Activity Investigation

Structure−activity investigation including design, syntheses, and evaluation of relative in vitro gene delivery efficacies of a novel series of cationic amphiphiles (1 − 10) containing mono-, di-, and trilysine headgroups are described in CHO, COS-1, and HepG2 cells. Several interesting and rather unexpected transfection profiles were observed. In general, lipid 1 with the myristyl tail used in combination with DOPE as colipid exhibited superior transfection properties compared to (a) the monolysinated analogues with longer hydrocarbon tails (lipids 2 − 4), (b) the dilysine (lipids 5 − 7) and the trilysine headgroup analogues (lipids 8 − 10), and (c) commercially available LipofectAmine with multiple positive charges in its polar region. As a preliminary estimate of the relative DNA-compacting efficacies of these new lysinated cationic lipids, the hydrodynamic diameters of representative lipoplexes were measured using dynamic laser light scattering technique. Our lipoplex size data are consistent with the notion that covalent grafting of an increasing number of positively charged functional groups in the headgroup region of cationic lipids need not necessarily result in more compacted lipoplexes. Both gel retardation and DNase I sensitivity assays indicated similar lipid/DNA binding interactions for all the novel mono-, di-, and trilysinated cationic lipids. MTT-assay-based cell viability results clearly demonstrate that the overall lower transfection properties of trilysine analogues (8 − 10) compared to their mono- (1-4) and dilysinated (5−7) counterparts are unlikely to originate from differential toxicity related effects. Taken together, the present findings support the notion that caution needs to be exercised in ensuring enhanced gene delivery efficacies of cationic lipids through covalent grafting of multiple lysine functionalities in the headgroup region.