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Cationic lipids have been effectively used as nonviral vectors for the delivery of polynucleic acids into the cytosol. In particular, lipids having hydroxy groups in the head group region facilitate the strong binding of polynucleic acids with the negatively charged phosphate groups of DNA via hydrogen bonding and electrostatic interactions and, thus regulate the transfection efficiency. In this regard, we designed and synthesised two cationic lipids, namely, aza sugar head group-based cationic lipids (Toc-Aza) and non-aza sugar-based cationic lipids (Toc-Pyr) as the control lipid. A well-known co-lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was used to formulate liposomes and lipoplexes, which were biophysically characterised for hydrodynamic diameters, global surface charges, and DNA binding. Three cell lines, namely, HEK-293, CHO, and HepG2 were used for cell viability assays and in vitro transfection studies. In vitro transfection studies revealed that the aza sugar head group-based cationic lipid (Toc-Aza) and the cationic lipid without hydroxyl groups on the aza sugar head group (pyrrolidine group) (Toc-Pyr) showed contrast transfection efficacies at a 2 : 1 charge ratio in all the three cell lines studied. Toc-Aza showed almost 10-fold better activities than Toc-Pyr at the 2 : 1 charge ratio in all the three cell lines studied. More importantly, the aza sugar head group-based cationic lipid (Toc-Aza) showed 1.5-fold transfection activity in HepG2 and HEK-293 cells and a similar pattern of transfection efficiency in CHO cell lines when compared with commercially available Lipofectamine 2000. These findings improve the knowledge of cationic liposomes and their effects on the variation in the hydroxyl functional group.

Understanding the role of structural units in cationic lipids used for gene delivery is essential in designing efficient gene delivery vehicles. Herein, we report a systematic structure–activity investigation on the influence of the spacer length on the DNA compaction ability and the transfection properties of gemini lipids with delocalizable cationic head groups. We have synthesized a series of dimeric cationic lipids varying in spacer length. The DNA binding interactions of liposomal formulations were characterized by gel electrophoresis and ethidium bromide (EtBr) exclusion assays. Condensation potentials were optimized and the best results were observed with cationic lipids possessing a 6 methylene spacer (TIM 6). We found that the size of the lipid/DNA complex decreased with the increase in spacer chain length up to a 6 methylene spacer TIM 6 and increased further. We have optimized the dimeric lipid/DOPE molar formulation using the β-galactosidase activity assay and found that the molar ratio of 1 : 1.5 (gemini lipid/DOPE) showed the maximum transfection among all molar ratios. The cellular uptake and co-localization of lipoplexes were observed by cell analysis and imaging using confocal microscopy. The results confirm that the lipoplex derived from lipid TIM 6 and pCMV-bgal/DNA internalizes via cellular endocytosis. The cytotoxicity studies using the MTT assay revealed that all formulations show comparable cell viability to the commercial standard even at higher charge ratios. Overall, the data suggest that the DNA compaction ability of these lipid dimers depends on the spacer chain length and the gemini lipid containing a six methylene aliphatic spacer has the maximum potential to deliver genes.

The incorporation of specific therapeutic gene into glioblastoma offers potent therapeutic strategy to treat the disease. Non-viral gene delivery vectors are of particular interest due to their tuneable transfection efficiency and easy scale-up. Herein, we demonstrate successful delivery of plasmid encoding tumor necrosis factor (TNF)-related apoptosis-inducing ligand (pTRAIL) using arginine-conjugated tocopherol lipid (AT) nanovesicles into glioblastoma cell lines. Another cationic lipid, glycine-conjugated tocopherol lipid (GT) having glycine in the head group region is also synthesized as a control lipid. Both lipid-derived liposomes effectively condensed the pDNA and the corresponding biomacromolecular assemblies (lipoplexes) are efficiently transfected into different cell lines. AT-based liposomes exhibit higher transfection efficacy in various cell lines, particularly selective in glioma cell lines. At an optimized N/P ratio, both the liposomal formulations show low cytotoxicity. AT-based lipoplexes have superior cellular uptake in U87 than the control lipid GT. The expression of TRAIL protein regulated death receptor and apoptosis signaling pathway is assayed by western blot using transfection of AT-based/pTRAIL into U87 cell lines. Induction of apoptosis in U87 cells exposed to AT-based/pTRAIL plasmid is evaluated by MTT assay as well as Annexin V-propidium iodide dual-staining assay. All results indicate that the developed AT-based/pTRAIL system offers a potentially safe and efficient therapeutic strategy for glioblastoma gene therapy.

Nonviral lipid-based vectors are promising transporting systems for the intracellular delivery of therapeutic gene sequences and directly influence the success of gene delivery. However, the associated drawbacks like lower transfection, toxicity, and targetability require further improvement. Thus, herein, we report a novel lipid formulation by the mixing of two distinct cationic surfactants such as tocopheryl succinate based cationic lipid and 1,12 dodecane based bolaamphiphile and prove it to be a good transfection reagent with its competing potential with the "golden standard", Lipofectamine 3000 (L3K). These interesting aggregations were named "Bolaliposome" and showed adequate unilamellar vesicle morphology under transmission electron microscopy, having a size of around 100 nm and could transfect efficiently different varieties of cell lines. Moreover, the generated complexes from bolaliposome and DNA (bolalipoplex) were characterized in terms of surface potential, hydrodynamic size, and gel electrophoresis. Various pharmacological inhibitors were also used in reporter gene expression to prove that the complexes followed the clathrin-mediated endocytosis. Finally, these findings would be helpful in the making of new aggregates and the development of better cytofectins. This was developed by optimizing the formulation based on the efficiency of reporter gene expression performed using the pEGFP-N3 plasmid.