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Fluorinated double-chain (poly)cationic lipids (one or both of these chains being ended by a highly fluorinated tail) which are close analogues of DOTMA, DMRIE or DPPES 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 5, these fluorinated cationic lipids condensed DNA, with or without the use of DOPE, to form fluorinated lipoplexes. No specific cell toxicity was evidenced for these new fluorinated lipoplexes. The efficiency of some of the fluorinated lipoplexes to transfect lung epithelial A549 cells was comparable to that of the first generation of fluorinated lipoplexes made from fluorinated analogues of DOGS (Transfectam) [Bioconjug. Chem. 12 (2001) 114]. These results, combined with the higher in vivo transfection potential found for fluorinated lipoplexes than for conventional lipoplexes or PEI polyplexes [J. Gene Med. 3 (2001) 109], confirm that fluorinated lipoplexes are very promising gene transfer systems.

The release of encapsulated carboxyfluorescein (CF) from liposomes made from various fluorinated amido-connected double-chain phosphocholines and their membrane permeability have been investigated at 37 degrees C in buffer and in human serum. These fluorinated membranes and liposomes display lower permeability coefficients and are able to retain more efficiently encapsulated CF than any of their respective conventional counterparts. Several of these liposomes are as effective as the first generation of liposomes based on fluorinated phosphatidylcholines, indicating that the chemical junction (ester/amide) and nature of the unit (glycerol, diaminopropanol, serine, ethanolamine) connecting the hydrophobic chains to the phosphocholine polar head have no significant effect on permeability and CF release. Our results show further that a fluorinated intramembrane layer reduces significantly the permeability of membranes in a liquid-crystalline state, protects the liposomes from the destabilizing effects of serum, and even increases their stability (in terms of dye retention) in serum when the membranes are in the gel state.

This paper is devoted to the morphological characterization, by freeze-fracture electron microscopy, and to the thermotropic phase behavior, by differential scanning calorimetry and X-ray diffraction of the aqueous dispersions of various fluorocarbon/fluorocarbon or mixed fluorocarbon/hydrocarbon 1,2- or 1,3-di-O-alkyl-glycerophosphocholines (PC) and 1,2-di-O-alkyl-glycerophosphoethanolamines (PE). The fluorinated PCs form classical lamellar phases and liposomes while an interdigitated lamellar phase has been evidenced for a hydrocarbon 1,3-analog. The fluorinated PEs display a lamellar to hexagonal phase transition which occurs almost simultaneously with the gel-to-fluid lamellar phase transition. The impact of each of the structural features [ether vs ester chemical junction connecting the hydrophobic chains on glycerol, their position (1,2- vs 1,3 isomers), number and length of the perfluoroalkylated chains, length of the fluorinated tail and hydrocarbon spacer, PC vs PE polar head] of the fluorinated phospholipids on the phase transition thermodynamic parameters (Tc, delta H, delta S) is discussed. Most of the liposomes formed from the fluorinated ether-PCs display a remarkable long-term shelf stability: they can be thermally sterilized and stored at room temperature for several months without any significant modification of their size and size distribution.

The stability (with respect to encapsulated carboxyfluorescein release) of liposomes made from various fluorocarbon 1,2-or 1,3-di-O-alkylglycerophosphocholines (ether-connected) and their membrane permeability have been investigated in buffer and in human serum. Membranes and liposomes, whether formulated with fluorocarbon/fluorocarbon or mixed fluorocarbon/hydrocarbon, 1,2- or 1,3-di-O-alkylglycerophospholipids, display lower permeability coefficients and are able to retain more efficiently encapsulated CF, even when incubated in human serum, than any of their conventional counterparts. These fluorinated liposomes are as stable as the first generation of liposomes formulated with their fluorocarbon ester-connected 1,2-di-O-acylglycerophosphocholine analogs. These results further confirm that a fluorinated intramembranar layer reduces the permeability of membranes (more significantly when they are in a fluid state), protects them from the destabilizing effects of serum components and increases even the stability of the fluorinated liposomes whose membranes are in the gel state when incubated in human serum. The impact of the modular structure of the fluorinated phospholipids (number of fluorocarbon chains, ether vs. ester bond, 1,2- vs. 1,3-isomer, etc...) and structure/permeability/ stability relationships are also presented.

The active encapsulation of doxorubicin (DOX) into fluorinated liposomes, the stability of these liposomes with respect to encapsulated DOX release in buffer and in human serum, and their H+/Na+ membrane permeability have been investigated and compared to those of their conventional hydrogenated analogues. These fluorinated liposomes are made from highly fluorinated phosphatidylcholines and contain a fluorinated core within their membrane. We found that the presence of this fluorinated core is not a barrier for the active encapsulation of DOX. Efficient (> 90%) and stable loading could be achieved using a transmembrane ammonium sulfate or even, in the absence of Na+, a transmembrane pH gradient. The higher H+/Na+ permeability found for the fluorinated membranes, as compared to conventional ones, is responsible for the lower stability observed for the DOX-loaded fluorinated liposomes when incubated in a physiological buffer (PBS) or in human serum. It is also noticeable that the retention of DOX is increased in human serum and for the liposomes whose membranes are in a gel or in a semi-fluid semi-gel state at 37 degrees C.

The stability (with respect to encapsulated carboxyfluorescein release) of fluorinated liposomes and their membrane permeability have been investigated in buffer and in human serum as compared to conventional hydrogenated analogues. These fluorinated liposomes are made from highly fluorinated phosphatidylcholines and contain a fluorinated core within their membrane. In buffer and in their fluid state, the fluorinated liposomes retain much more efficiently their entrapped content and display lower membrane permeability coefficients than any of their hydrogenated counterparts. This indicates that the fluorinated core acts as a very efficient barrier to permeation. In terms of molecular structure/permeability relationships, the thicker the fluorinated lipophobic core, the more efficient the barrier to permeation. In their gel state, the fluorinated core has, however, almost no effect on permeation. Interestingly, some of the 'fluid' fluorinated liposomes were even less permeable than 'gel' or 'gel-like' ones, including egg phosphatidylcholines/cholesterol liposomes. Human serum destabilizes the 'fluid' fluorinated liposomes but to a lesser extent than the 'fluid' hydrogenated ones, indicating that the fluorinated lipophobic core inside the liposomal membrane protects the vesicles, possibly by reducing their interactions with serum components. 'Gel' or 'gel-like' fluorinated liposomes are significantly more stable in serum than in buffer. They are also more stable than conventional 'gel' or 'gel-like' liposomes.

The polymorphic phase behavior of the F-alkyl modified phosphatidylcholines FnCmPC with Fn = CnF2n + 1 and Cm = -(CH2)m- and the physicochemical properties of their aqueous dispersions have been investigated. We show that the supramolecular assemblies formed by F4C4PC, F6C4PC, F8C4PC and F4C10PC dispersed in water consist of liposomes. F6C10PC forms, as does F8C10PC, a ribbon-like phase (two-dimensional centered rectangular lattice) at 25 degrees C, but on heating, it forms a lamellar phase. Upon cooling, the lamellar gel phase is metastable and converts slowly back into the ribbon-like phase. Analyses of the dispersions before and after heat sterilization and upon storage at 25 degrees C reveal an exceptional stability of the FnCmPC-based liposomes which contrasts strongly with that of DPPC vesicles. This enhanced stability most likely arises from the increased hydrophobic character resulting from the presence of the perfluoroalkyl tails. The gel to fluid phase transition temperature of the FnCmPCs is found to be related to the total length of the hydrophobic chain and more markedly to the length of the perfluoroalkyl tail. This phase transition is first induced by the melting of the fluorocarbon chain. Each portion of the Fn tail and of the hydrocarbon spacer experiences intrinsic changes of molecular motion with temperature. The partitioning of a lipophilic/hydrophilic paramagnetic probe between the aqueous and lipidic phases present in the FnCmPC dispersions shows that an increase in fluorophilic character results in a lower solubility of the probe in the membrane, thus reflecting a dramatic decrease of the membrane's lipophilicity.