title: Efficacious gene silencing in serum and significant apoptotic activity induction by survivin downregulation mediated by new cationic gemini tocopheryl lipids.
abstract: Nonviral gene delivery offers cationic liposomes as promising instruments for the delivery of double-stranded RNA (ds RNA) molecules for successful sequence-specific gene silencing (RNA interference). The efficient delivery of siRNA (small interfering RNA) to cells while avoiding unexpected side effects is an important prerequisite for the exploitation of the power of this excellent tool. We present here six new tocopherol based cationic gemini lipids, which induce substantial gene knockdown without any obvious cytotoxicity. All the efficient coliposomal formulations derived from each of these geminis and a helper lipid, dioleoylphosphatidylethanolamine (DOPE), were well characterized using physical methods such as atomic force microscopy (AFM) and dynamic light scattering (DLS). Zeta potential measurements were conducted to estimate the surface charge of these formulations. Flow cytometric analysis showed that the optimized coliposomal formulations could transfect anti-GFP siRNA efficiently in three different GFP expressing cell lines, viz., HEK 293T, HeLa, and Caco-2, significantly better than a potent commercial standard Lipofectamine 2000 (L2K) both in the absence and in the presence of serum (FBS). Notably, the knockdown activity of coliposomes of gemini lipids was not affected even in the presence of serum (10% and 50% FBS) while it dropped down for L2K significantly. Observations under a fluorescence microscope, RT-PCR, and Western blot analysis substantiated the flow cytometry results. The efficient cellular entry of labeled siRNA in GFP expressing cells as evidenced from confocal microscopy put forward these gemini lipids among the potent lipidic carriers for siRNA. The efficient transfection capabilities were also profiled in a more relevant fashion while performing siRNA transfections against survivin (an anti-apoptotic protein) which induced substantial apoptosis. Furthermore, the survivin downregulation improved the therapeutic efficacy levels of an anticancer drug, doxorubicin, significantly. In short, the new tocopherol based gemini lipids appear to be highly promising for achieving siRNA mediated gene knockdown in various cell lines.
authors.affiliation: Department of Organic Chemistry, Indian Institute of Science , Bangalore 560012, India.
authors.forename: Krishan
authors.initials: K
authors.lastname: Kumar
authors.forename: Bappa
authors.initials: B
authors.lastname: Maiti
authors.forename: Paturu
authors.initials: P
authors.lastname: Kondaiah
authors.forename: Santanu
authors.initials: S
authors.lastname: Bhattacharya
mesh_terms: D017209:Apoptosis / Q000235:genetics*; D015153:Blotting, Western; D018938:Caco-2 Cells; D002460:Cell Line; D024202:Electrophoretic Mobility Shift Assay; D020868:Gene Silencing / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000097:blood / Q000235:genetics*; D008055:Lipids / Q000502:physiology*; D008081:Liposomes / Q000737:chemistry; D018625:Microscopy, Atomic Force; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000077022:Survivin; D014162:Transfection
chemical_list: C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D008055:Lipids; D008081:Liposomes; D034741:RNA, Small Interfering; D000077022:Survivin
keywords: anti-GFP siRNA; gemini lipids; knockdown; serum; survivin; transfection