Synthesis and Transfection Efficiency of Cationic Oligopeptide Lipids: Role of Linker

Abstract

In the design of new cationic lipids for gene transfection, the chemistry of linkers is widely investigated from the viewpoint of biodegradation and less from their contribution to the biophysical properties. We synthesized two dodecyl lipids with glutamide as the backbone and two lysines to provide the cationic headgroup. Lipid 1 differs from Lipid 2 by the presence of an amide linkage instead of an ester linkage that characterizes Lipid 2. The transfection efficiency of lipoplexes with cholesterol as colipid was found to be very high with Lipid 1 on Chinese Hamster Ovary (CHO) and HepG2 cell lines, whereas Lipid 2 has shown partial transfection efficiency on HepG2 cells. Lipid 1 was found to be stable in the presence of serum when tested in HepG2 and CHO cells albeit with lower activity. Fluorescence-based dye-binding and agarose gel-based assays indicated that Lipid 1 binds to DNA more efficiently than Lipid 2 at charge ratios of >1:1. The uptake of oligonucleotides with Lipid 1 was higher than Lipid 2 as revealed by confocal microscopy. Transmission electron microscopy (TEM) images reveal distinct formation of liposomes and lipoplexes with Lipid 1 but fragmented and unordered structures with Lipid 2. Fusion of Lipids 1 and 2 with anionic vesicles, with composition similar to plasma membrane, suggests that fusion of Lipid 2 was very rapid and unlike a fusion event, whereas the fusion kinetics of Lipid 1 vesicles was more defined. Differential scanning calorimetry (DSC) revealed a high T(m) for Lipid 1 (65.4 °C) while Lipid 2 had a T(m) of 23.5 °C. Surface area-pressure isotherms of Lipid 1 was less compressible compared to Lipid 2. However, microviscosity measured using 1,6-diphenyl-1,3,5-hexatriene (DPH) revealed identical values for vesicles made with either of the lipids. The presence of amide linker apparently resulted in stable vesicle formation, higher melting temperature, and low compressibility, while retaining the membrane fluid properties suggesting that the intermolecular hydrogen bonds of Lipid 1 yielded stable lipoplexes of high transfection efficiency.

Post-Transcriptional Regulation of the GASC1 Oncogene with Active Tumor-Targeted siRNA-Nanoparticles

“…48,49 In our study, we used two structurally different conjugates (Tf–PEI and Tf-PEG-PEI) which were comparable in physicochemical properties but different in transfection, binding, and uptake efficiencies. As a result, the efficiency of PEI conjugates strongly depends on the linker chemistry.…”

Cationic lipids for gene delivery: many players, one goal

“…In this light, Vacus and co-workers suggested that the ability of amide linkers to form intermolecular hydrogen bonding is responsible for the high melting temperature of the lipids and for the lipoplex stability (Boukhnikachvili et al, 1997). Such speculations were later confirmed by Gopal et al, who demonstrated that amide-bearing cationic lipids were much more stable and effective in transfection than ester-tethered transfectants (Gopal et al, 2011).…”

Novel 1,2,3-triazolium-based dicationic amphiphiles synthesized using click-chemistry approach for efficient plasmid delivery

“…This was followed by the addition of complexes to the wells in a triplicate manner, and the cells were allowed to incubate in the CO 2 incubator for about 4 h. The complex media were replaced by 10% serum media (0.3 mL per well) and the incubation was continued for about 48 h in a CO 2 incubator and assayed by following the protocol, as mentioned earlier. 46 Furthermore, to support the transfection patterns obtained from beta gal transfection, one more transfection was performed using the pEGFP-N 3 plasmid in HEK-293 and CHO cells. Briefly, the cells were seeded onto a 12-well plate at a density of 4 × 10 4 cells per well a day before the transfection.…”