Design, syntheses, and transfection biology of novel non-cholesterol-based guanidinylated cationic lipids.

The design of efficacious cationic transfection lipids with guanidinium headgroups is an actively pursued area of research in nonviral gene delivery. Herein, we report on the design, syntheses, and gene transfection properties of six novel non-cholesterol-based cationic amphiphiles (1-6) with a single guanidinium headgroup in transfecting CHO, COS-1, MCF-7, A549, and HepG2 cells. The in vitro gene transfer efficiencies of lipids 1-6 were evaluated using both the reporter gene and the whole cell histochemical X-gal staining assays. The efficiencies of lipids 1-3, in particular, were found to be about 2- to 4-fold higher than that of commercially available LipofectAmine in transfecting COS-1, CHO, A-549, and MCF-7 cells. However, the relative transfection efficiencies of lipids 1-3 and LipofectAmine were found to be comparable in HepG2 cells. Cholesterol was found to be a more efficacious co-lipid than dioleoyllphosphatidyl ethanolamine (DOPE). In general, lipids 1-3 containing the additional quaternized centers were observed to be more transfection efficient than lipids 4-6 with less positive headgroups. MTT-assay-based cell viability measurements in representative CHO cells revealed high (>75%) cell viabilities of lipids 1-6 across the lipid/DNA charge ratios 0.1:1 to 3:1. Electrophoretic gel patterns observed in DNase I protection experiments support the notion that enhanced degradation of DNA associated with lipoplexes of lipids 4-6 might play some role in diminishing their in vitro gene transfer efficacies. Size and global surface charge measurement by a dynamic laser light scattering instrument equipped with zeta-sizing capacity revealed the nanosizes and surface potentials of both the transfection efficient and the incompetent lipoplexes to be within the range of 200-600 nm and +3.4 to -34 mV, respectively. To summarize, given the feasibility of a wide range of structural manipulations in the headgroup regions of non-cholesterol-based cationic amphiphiles, our present findings are expected to broaden the potential of cationic amphiphiles with guanidinium headgroups for use in nonviral gene therapy.