Improved in vitro gene transfer mediated by fluorinated lipoplexes in the presence of a bite salt surfactant

Background Progress in the field of gene transfer with non-viral vectors re quires systems that allow efficient gene expression in the presence of biol ogical fluids such as pulmonary surfactants, for gene transfer to the respi ratory epithelium for cystic fibrosis gene therapy, or bile salts (which co ntain powerful anionic detergents), for gene transfer to the biliary epithe lium for gene therapy of the hepatobiliary disease associated with cystic f ibrosis (CF). We have performed a comparative analysis of the disintegratio n and DNA accessibility of fluorinated and conventional lipoplexes, and the ir in vitro transfection potential in the presence of a powerful biliary su rfactant. Methods The disintegration and DNA accessibility of conventional and fluori nated cationic lipoplexes and their in vitro transfection efficiency of hum an lung carcinoma epithelial A549 cells were studied in the presence of var ious concentrations of sodium taurocholate (STC), an anionic bile salt dete rgent. The conventional and fluorinated lipoplexes were formulated from Tra nsfectam(R) (or DOGS) and from fluorinated lipospermines, analogs of DOGS, respectively, and a luciferase reporter plasmid. The fluorinated lipids use d in the present study were selected for their different degrees of fluorin ation in order to investigate the impact on stability and transfection. The effects of the detergent on lipoplex integrity were examined by evaluating the ability of the lipospermines to prevent, in the presence of the surfac tant, ethidium bromide (BET) intercalation into the plasmid (fluorescence m onitoring). Results Fluorinated cationic lipoplexes exhibited greater stability than DO GS lipoplexes with respect to STC lyric activity. Indeed, while the DOGS li poplexes were fully disintegrated at a [STC]/[lipid] molar ratio of 1320, a ll the DNA intercalation sites of the most fluorinated lipoplexes investiga ted became accessible to BET for a two-fold higher [STC]/[Iipid] molar rati o. A higher transfection potential in the presence of the detergent was als o shown the fluorinated lipoplexes as compared with the DOGS preparation. A t a 10 mM concentration of STC and at a [STC]/[lipid] molar ratio of 264, w hen mediated by DOGS was fully inhibited while the detergent no inhibitory effects on the lipofection mediated by the fluorinated DF4C11-GS [spermine- 5-carboxyglycine N,N-di-11(F-butyl)-undecylamide] or DF6E11-GS {spermine-5- carboxyglycine N,N-di-[11-(F-hexyl)-undec-10-enyl]amide} lipospermines. A h igher detergent concentration (up to 17.5 mM) and a higher [STC]/[lipid] ra tio (up to 462) were necessary to inhibit lipofection by the fluorinated fo rmulations. Overall, the lipoplex stability and transfection potential in t he presence of the detergent was found to improve with increasing degrees o f fluorination of the lipospermines. Conclusions The present work shows improved stability of, and higher lipofe ction levels with, fluorinated lipoplexes in the presence of surfactants. T he results confirm the very promising potential of fluorinated lipoplexes a s gene transfer vectors. These compounds constitute a very attractive alter native to their more conventional homologs. The correlation found between t he degree of fluorination of the lipoplexes, their stability and their lipo fection levels suggests that enhanced lipophobic and hydrophobic properties protect them against disintegration and, consequently, prevents DNA from b eing degraded and from interacting with lipophilic and hydrophilic biocompo unds responsible for lipofection inhibition. Copyright (C) 2001 John Wiley & Sons, Ltd.