A cationic cytofectin with long spacer mediates favourable transfection in transformed human epithelial cells

The synthesis and transfection potential of a novel cationic cholesterol cytofectin with a dimethylamino head group and a long 12 atom, 15 angstrom spacer incorporating relatively polar amido and dicarbonyl hydrazine linkages are reported. Thus N,N-dimethylaminopropylamidosuccinylcholesterylformylhydrazide (MS09) in equimolar admixture with dioleoylphosphatidylethanolamine (DOPE) forms stable unilamellar liposomes (80-150 nm) which cluster into very effective transfecting, serum nuclease-resistant, lipoplexes with DNA (180-200 nm) at a liposome+/DNA- molar charge ratio of 2.8:1 (12:1, w/w). Gel retardation and ethidium displacement assays confirmed that DNA was fully liposome-associated and maximally compacted at this ratio. Transfection levels in three human transformed epithelia] cell lines, as established by luciferase transgene activity, was found to be optimal at this charge ratio and in the following order: cervical carcinoma (HeLa) > oesophageal carcinoma (SNO) > hepatoblastorna (HepG2). Activity in the murine fibroblast line NIH-3T3 was comparable to that in HepG2 cells. MS09 lipoplexes achieved approximately three-times and two-times greater activity than Lipofectin((R)) complexes in HeLa and SNO cells, respectively, whilst comparable levels were recorded in HepG2 and NIH-3T3 cells. MS09 lipoplexes were well tolerated by HepG2, HeLa and SNO cells with cell numbers found to be 80, 85 and 75% of untreated cultures, respectively, at the optimal transfection concentration. These lipoplexes also exhibited high activity in the presence of 10% foetal bovine serum (FBS) in HeLa (17% inhibition) and HepG2 (33% inhibition) cells. (c) 2005 Elsevier B.V. All rights reserved.