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There is a need for the development of nonviral gene transfer systems with improved and original properties. "Fluorinated" lipoplexes are such candidates, as supported by the remarkably higher in vitro and in vivo transfection potency found for such fluorinated lipoplexes as compared with conventional ones or even with PEI-based polyplexes (Boussif, O., Gaucheron, J., Boulanger, C., Santaella, C., Kolbe, H. V. J., Vierling, P. (2001) Enhanced in vitro and in vivo cationic lipid-mediated gene delivery with a fluorinated glycerophosphoethanolamine helper lipid. J. Gene Med. 3, 109-114). Here, we describe the synthesis of fluorinated glycerophosphoethanolamines (F-PEs), close analogues of dioleoylphosphatidylethanolamine (DOPE), and report on their lipid helper properties vs that of DOPE, as in vitro gene transfer components of fluorinated lipoplexes based on pcTG90, DOGS (Transfectam), or DOTAP. To evaluate the contribution of the F-PEs to in vitro lipoplex-mediated gene transfer, we examined the effect of including the F-PEs in lipoplexes formulated with these cationic lipids (CL) for various CL:DOPE:F-PE molar ratios [1:(1 - x):x with x = 0, 0.5 and 1; 1:(2 - y):y with y = 0, 1, 1.5, and 2], and various N/P ratios (from 10 to 0.8, N = number of CL amines, P = number of DNA phosphates). Irrespective of the F-PE chemical structure, of the colipid F-PE:DOPE composition, and of the N/P ratio, comparable transfection levels to those of their respective control DOPE lipoplexes were most frequently obtained when using one of the F-PEs as colipid of DOGS, pcTG90, or DOTAP in place of part of or of all DOPE. However, a large proportion of DOGS-based lipoplexes were found to display a higher transfection efficiency when formulated with the F-PEs rather than with DOPE alone while the opposite tendency was evidenced for the DOTAP-based lipoplexes. The present work indicates that "fluorinated" lipoplexes formulated with fluorinated helper lipids and conventional cationic lipids are very attractive candidates for gene delivery. It confirms further that lipophobicity and restricted miscibility of the lipoplex lipids with the endogenous lipids does not preclude efficient gene transfer and expression. Their transfection potency is rather attributable to their unique lipophobic and hydrophobic character (resulting from the formulation of DNA with fluorinated lipids), thus preventing to some extent DNA from interactions with lipophilic and hydrophilic biocompounds, and from degradation.

Background: Progress in the field of gene transfer with non-viral vectors requires systems that allow efficient gene expression in the presence of biological fluids such as pulmonary surfactants, for gene transfer to the respiratory epithelium for cystic fibrosis gene therapy, or bile salts (which contain powerful anionic detergents), for gene transfer to the biliary epithelium for gene therapy of the hepatobiliary disease associated with cystic fibrosis (CF). We have performed a comparative analysis of the disintegration and DNA accessibility of fluorinated and conventional lipoplexes, and their in vitro transfection potential in the presence of a powerful biliary surfactant., Methods: The disintegration and DNA accessibility of conventional and fluorinated cationic lipoplexes and their in vitro transfection efficiency of human lung carcinoma epithelial A549 cells were studied in the presence of various concentrations of sodium taurocholate (STC), an anionic bile salt detergent. The conventional and fluorinated lipoplexes were formulated from Transfectam" (or DOGS) and from fluorinated lipospermines, analogs of DOGS, respectively, and a luciferase reporter plasmid. The fluorinated lipids used in the present study were selected for their different degrees of fluorination 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 surfactant, ethidium bromide (BET) intercalation into the plasmid (fluorescence monitoring)., Results: Fluorinated cationic lipoplexes exhibited greater stability than DOGS lipoplexes with respect to STC lytic activity. Indeed, while the DOGS lipoplexes were fully disintegrated at a [STC]/[lipid] molar ratio of 1,320, all the DNA intercalation sites of the most fluorinated lipoplexes investigated became accessible to BET for a two-fold higher [STC]/[lipid] molar ratio. A higher transfection potential in the presence of the detergent was also shown for the fluorinated lipoplexes as compared with the DOGS preparation. At a 10 mM concentration of STC and at a [STC]/[lipid] molar ratio of 264, lipofection when mediated by DOGS was fully inhibited while the detergent had 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 higher detergent concentration (up to 17.5 mM) and a higher [STC]/[lipid] ratio (up to 462) were necessary to inhibit lipofection by the fluorinated formulations. Overall, the lipoplex stability and transfection potential in the presence of the detergent was found to improve with increasing degrees of fluorination of the lipospermines., Conclusions: The present work shows improved stability of, and higher lipofection levels with, fluorinated lipoplexes in the presence of surfactants. The results confirm the very promising potential of fluorinated lipoplexes as gene transfer vectors. These compounds constitute a very attractive alternative to their more conventional homologs. The correlation found between the degree of fluorination of the lipoplexes, their stability and their lipofection levels suggests that enhanced lipophobic and hydrophobic properties protect them against disintegration and, consequently, prevents DNA from being degraded and from interacting with lipophilic and hydrophilic biocompounds responsible for lipofection inhibition.

Background: One of the main drawbacks of synthetic, non-viral gene vectors is their relatively low in vivo efficiency when compared with viral vectors. The present paper describes the use of a partially fluorinated glycerophosphoethanolamine (F-PE), a close analog of DOPE, which, as a helper lipid with the cationic lipopolyamine pcTG90, increases its in vitro and in vivo gene transfer capability to a larger extent than DOPE., Methods: To evaluate the contribution of F-PE to lipoplex-mediated gene transfer, the effect of including F-PE in lipoplexes formulated with the lipopolyamine pcTG90 for various pcTG90/DOPE/F-PE molar ratios [1:(1-x): x; 1:(2-y):y] was examined. For the in vitro analyses on human lung carcinoma epithelial A549 cells, the lipoplexes were formulated with the luciferase reporter plasmid pTG11033 using various N/P ratios (from 10 to 0.8, N = number of pcTG90 amines, P = number of DNA phosphates). The in vivo analyses were performed (1) with the luciferase reporter plasmid pCMV-Luc, which gives higher luciferase expression in the lung than pcTG11033; (2) with pcTG90/co-lipid(s) (1:2) lipoplexes which yield higher expression than the (1:1) formulations; and (3) by intravenous (iv) injection into the tail vein of mice., Results: The efficiency of the F-PE lipoplexes to transfect in vitro A549 cells was significantly higher (5-90-fold) than that of DOPE lipoplexes, when formulated in HEPES. However, when formulated in 5% glucose, both co-lipids display a comparable transfection helper potential. Most remarkably, an up to eight-fold increase of luciferase expression could be measured in the lung after iv injection of pcTG90/F-PE (1:2) N/P 5 lipoplexes as compared with the pcTG90/DOPE lipoplexes. It led also to higher luciferase expression than PEI(ExGen500)/pCMV-Luc N/P 10 polyplexes. Besides expression in lung, low levels of luciferase expression were also observed in heart, spleen and liver., Conclusion: The present work, showing a higher in vitro and in vivo transfection potential for lipoplexes formulated with a partially fluorinated co-lipid as compared with its analogous DOPE lipoplexes or PEI polyplexes, indicates that 'fluorinated' lipoplexes are attractive candidates for in vivo applications.

Fluorinated double-chain lipospermines (one or both of these chains being ended by a highly fluorinated tail of various length) which are close analogues of DOGS (Transfectam) were designed as synthetic vectors for gene delivery. For N/P ratios (N = number of amine functions of the lipid; P = number of DNA phosphates) from 0.8 to 10, these lipospermines condensed DNA, with or without the use of DOPE, to form fluorinated lipoplexes. The efficiency of the fluorinated lipoplexes to transfect lung epithelial A549 cells was significantly higher than that of the DOGS lipoplexes. No specific cell toxicity was evidenced for the fluorinated lipoplexes as compared to that of the DOGS ones. The palette of structural elements explored allowed to determine those required for efficient transfection, highlighting the importance of highly fluorinated chains, the unique properties of unsaturated double-chain lipids and of the use of DOPE as helper lipid on transfection.

We report on the synthesis of a series of lipopolyamine telomers I-14,n, I-18,n, and II-18,n and on their in vitro gene-transfer capability. Their structure consists of a polyamine polar moiety, including n primary amine functions (from 1 to 70), connected to a hydrophobic moiety, including two hydrocarbon C14 or C18 chains, through a mercaptopropanoyl or mercaptoglyceryl unit and an amide or ether function. They were obtained by telomerization of N-[2-[(BOC)aminoethyl]]acrylamide with N,N-ditetradecyl- and N,N-dioctadecylpropanamide-3-thiol and rac-1,2-dioctadecyloxypropane-3-thiol, respectively, then BOC deprotection. For N/P ratios (N = number of telomer amine equivalents; P = number of DNA phosphates) from 0.8 to 10, these lipopolyamines condensed DNA, with or without the use of DOPE, forming lipopolyplexes or teloplexes of mean sizes less than 200 nm. Some trends, structure-activity and structure-toxicity relationships, were established to achieve both highest in vitro transfection levels and cell viability. Thus, DNA formulations based on telomers I-14,20 and I-18,20 and for N/P ratios lower than 5 led to the most efficient teloplex formulations for plasmid delivery to lung epithelial A549 cells.