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

Abstract

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.

An intracellular lamellar–nonlamellar phase transition rationalizes the superior performance of some cationic lipid transfection agents

“…Lipoplex size has been suggested to modulate transfection activity, with larger (within limits) lipoplexes being generally more efficient (27,28). Recent experiments showed, however, that lipoplex size by itself does not necessarily directly correlate to transfection efficiency (29,30), and examples now exist in which smaller lipoplexes are more efficient than larger ones (21,31). Therefore, we examined the aggregate sizes of the two C18͞C10-EPC compounds and their lipoplexes.…”

Cationic liposomes as non‐viral carriers of gene medicines: Resolved issues, open questions, and future promises

“…To this end, recently we have designed and synthesized a series of novel cationic lipids with mono-, di-, and tri-lysine head-groups and common hydrophobic anchors. 80 The in vitro gene delivery efficacies of these lipids were compared in CHO, COS-1, and HepG2 cells. This structure-activity investigation revealed that the cationic lipid with a mono-lysine head-group and myristyl tail (DTMLAC, Fig.…”

On the disulfide‐linker strategy for designing efficacious cationic transfection lipids: an unexpected transfection profile

“…1) was designed and synthesized in our on-going structure-activity program in the area of liposomal gene delivery [25][26][27][28][29][30] as a non-glycerol based mono-cationic lipid where the positively charged terminal amine group was separated from the hydrophobic aliphatic tails by a reasonably long spacer arm. Fig.…”

“…Such complex transfection pathway makes rational design of efficient cationic transfection lipids an arduous task. A number of recent structure-activity investigations [21][22][23][24], including our own [25][26][27][28][29][30], have thrown significant new insights into the various architectural elements of cationic lipids necessary for overcoming the above mentioned cellular barriers involved in lipofection process.…”