Spacer-arm modulated gene delivery efficacy of novel cationic glycolipids: design, synthesis, and in vitro transfection biology.

Design, syntheses and relative in vitro gene delivery efficacies of six novel cationic glycolipids 1-6 containing open-form galactosyl units in CHO, COS-1, MCF-7 and A549 cells are described. The results of the present structure-activity investigation convincingly demonstrate that the in vitro gene delivery efficacies of galactosylated cationic glycolipids are strikingly dependent on the absence of a spacer-arm between the open-form galactose and the positively charged nitrogen atom in their headgroup region. While the cationic glycolipids 1-3 with no headgroup spacer unit between the positively charged nitrogen and galactose showed high in vitro gene transfer efficacies in all four cells (lipids 1 and 2 with myristyl and palmityl tails, respectively, being the most efficacious), lipids 4-6 with five-carbon spacer units between the quaternized nitrogen and galactose heads were essentially transfection incompetent. The transfection inhibiting role of the five-carbon spacer unit in the headgroup region of the present novel class of cationic lipids was demonstrated by both beta-galactosidase reporter gene expression and histochemical X-gal staining assays. Results of MTT assay-based cell viability measurements in representative MCF7 cells show that cell viabilities of lipoplexes (lipid:DNA complexes) prepared from all the lipids 1-6 are remarkably high. Thus, possibilities of differential cellular cytotoxicities playing any key role behind the strikingly contrasting transfection properties of lipids 1-3 with no spacer and lipids 4-6 with a spacer unit in the headgroup regions was ruled out. Electrophoresis gel patterns in DNase I sensitivity assays are consistent with more free DNA (accessible to DNase I) being present in lipoplexes of lipids 4-6 than in lipoplexes of lipids 1-3. Thus, the results of our DNase I protection experiments support the notion that enhanced degradation of DNA associated with lipoplexes of lipids 4-6 may play an important role in abolishing their in vitro gene transfer efficacies.

Distance of hydroxyl functionality from the quaternized center influence DNA binding and in vitro gene delivery efficacies of cationic lipids with hydroxyalkyl headgroups.

In vitro gene delivery efficacies of cationic amphiphiles 1-7 (Scheme 1) were measured by both the reporter gene expression assays in CHO, COS-1, HepG2, and MCF7 cells and by the whole cell histochemical X-gal staining of representative Chinese hamster ovary cells. Our results demonstrated that in vitro gene delivery efficiencies of cationic lipids with hydroxyalkyl headgroups are adversely affected by increased covalent distances between the hydroxyl functionality and the cationic centers. Findings in the DNase I protection experiments and transmission electron microscopic study support the notion that such compromised gene delivery efficacies may originate from poor lipid-DNA binding interactions and significantly increased lipoplex nanosizes.

Cationic amphiphile with shikimic acid headgroup shows more systemic promise than its mannosyl analogue as DNA vaccine carrier in dendritic cell based genetic immunization.

Mannosylated cationic vectors have been previously used for delivering DNA vaccines to antigen presenting cells (APCs) via mannose receptors expressed on the cell surface of APCs. Here we show that cationic amphiphiles containing mannose-mimicking quinic acid and shikimic acid headgroups deliver genes to APCs via mannose receptor. Cationic amphiphile with shikimic acid headgroup was more efficacious than its mannosyl counterpart in combating mouse tumor growth by dendritic cell (the most professional APC) based genetic immunization.