Two new N(a),N(b)-Bis[3-(trimethylamino)propyl]-perfluoroalkanes diamide-Iodide with (a=1; b=10) and (a=1; b=12), differing by the fluorocarbon tail spacer (8 or 10 CF2), and the N(1),N(12)-Bis[3-(trimethylamino)propyl]-dodecanediamide-Iodide have been synthesized in two steps involving the appropriate dimethyl perfluoro or hydrogeno alkane dicarboxylate, N,N-dimethyl-1,3-propane diamine, and methyl iodide. Their surface properties were studied at 45°C in aqueous solutions using conductivity, surface tension, and dynamic light scattering (DLS) measurements. A comparison of the micellar parameters usually obtained by these techniques has been carried out. The critical micelle concentrations (CMCs), the geometric packing parameters, the surface excess concentration, the area/surfactant molecule at the interface, the micellar ionization degree β and the free energies of micellization, the diffusion coefficient, and the hydrodynamic diameter were discussed. The effect of added KI on these parameters and on the size and on the kind of aggregates has been also investigated. The addition of 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) to the surfactant solutions, where the size of the complex DOPE-surfactant was determined by DLS over a wide range of concentrations, reveals a very different behavior comparing fluorinated and hydrogenated compounds.

Structural modifications of unsaturated sodium carboxylate surfactants in terms of trifluoromethylation associated with the hydrocarbon chain length have been studied, the synthesis is described, and aggregation properties have been examined by conductimetry and vapor pressure osmometry between 30°C and 45°C. No strong effect of adding a CF3 group was observed on the Critical Micellar Concentrations. However, the thermodynamic study shows the specific effect exerted by the CF3 group through the enhancement of the entropic contribution.

A new series of N-[3-(trimethylamino)propyl]-perfluoro-N-glucosyl amide-Iodides, differing by the length of the fluorocarbon tail (7, 9, and 11), have been synthesized in three steps involving unprotected glucose, N,N-dimethyl-1,3-propane diamine, the appropriate methyl-perfluoroalcanoate, and methyl iodide. Their aggregation and surface properties were studied in aqueous solution using conductivity, surface tension, and dynamic light scattering measurements. The critical micelle concentrations (CMC), the micellar aggregation numbers, the geometric packing parameters, the area/surfactant molecule at the interface, the surface excess concentration, the micellar ionization degree β, and the free energies of micellization have been investigated. DLS results show various morphologies of aggregates such as micelles and vesicles according to the increase in the hydrophobic chain length.

In order to promote siRNA transfer in tumour cells, we used an original cationic lipid, synthesized in our laboratory, dimethyl-hydroxyethyl-aminopropane-carbamoyl-cholesterol (DMHAPC-Chol). Liposomes were prepared from this lipid and dioleoylphosphatidylethanolamine (DOPE) in equimolar proportion. Its transfecting capacity was evaluated using ELISA, cell cytometry, and RT-PCR in estimating the silencing effect of VEGF siRNA. This liposome efficiently delivered VEGF siRNA in two human cancer cell lines abundantly secreting VEGF, A431 and MDA-MB-231. Results showed that 50 nM of VEGF siRNA carried by DMHAPC-Chol/DOPE liposomes already silenced more than 90% of VEGF in these cells. A comparative study with two commercial carriers indicated that the inhibition induced by VEGF siRNA transported by cationic DMHAPC-Chol/DOPE liposomes was comparable to that induced by INTERFERin and better than lipofectamine 2000. Moreover, a transfection by a GFP plasmid followed by a GFP siRNA showed that DMHAPC-Chol/DOPE liposomes compared to lipofectamine were less efficient for plasmid but better for siRNA transport. Following one of our previous works concerning cell delivery of plasmid ( Percot et al., 2004 ), the main interest of results presented here resides in the double potential of DMHAPC-Chol/DOPE liposomes to deliver little-sized siRNA as well as large nucleic acids in cells.

A series of perfluorinated cationic surfactants and their corresponding hydrocarbon ones whose general formula is CnX2n+1-C(O)NH-(CH2)(3)-N+Me3,I-, with X = F, H and n = 9, 11, have been synthesized via two steps. Their aggregative and surface-active properties were studied in aqueous solution using tensiometry and conductimetry. The critical micelle concentrations and the molecular areas at the air/water interface of fluorinated surfactants are lower than those of their hydrocarbon homologues. Micellar aggregation numbers and geometric packing parameters have been investigated. The results indicated that fluorocarbon surfactants tend to form lamellar aggregates while the hydrocarbon ones associate into spherical aggregates. (C) 2010 Elsevier B.V. All rights reserved.

Interactions in aqueous solution between polyvinyl alcohol (PVA) and various short chain nonionic polyols surfactants having six to nine carbon atoms and two to three hydroxyl groups are investigated using tensiometry, viscosity, and dynamic laser light scattering techniques. Despite the fact that weak interactions are noticed, they begin to occur at surfactant concentrations far lower than the Critical Micellar Concentration. Partition coefficients of the surfactants between water and the PVA macromolecules are determined, and the contributions of the surfactant alkyl chain length on one hand and of the hydroxyl groups on the other hand to the PVA interactions with monomer surfactants are discussed in terms of thermodynamic contributions.

Cationic liposomes constitute a new class of bilayer, which are able to form complexes with nucleic acids (DNA or RNA), help to cross the cellular membranes, and then deliver them into cells. This interesting property opens several applications for cationic liposomes, in particular in gene therapy, vaccination and biotechnology. The aim of this chapter is to review the structural properties of these liposomes and to describe the methods of characterization as well as methods to observe their pathway in cells after the passage through the cellular barrier. After the description of chemical structures of cholesterol-based cationic lipids investigated in our laboratory, their formation into liposomes, their interaction with nucleic acids and the internalization of complexes in cells will be dealt with. The cytotoxicity and the transfection level are outlined, and applications of cationic liposomes for transfer of interest DNA are illustrated.

In this paper, liposomes containing a lipopeptide bearing a ligand specifically recognized by neuropilin-1 (NRP-1) have been used to target a human breast cancer cell line overexpressing this receptor. The synthesis of this lipopeptide, C16-A7R, formed by the sequence of 7 aminoacids ATWLPPR, linked to a palmitoyl fatty chain by an amide bond was described. After the characterisation of cationic liposomes formulated with the lipopeptide, the results obtained using various techniques showed that the lipopeptide-based liposomes were well accumulated in cells of the human breast cancer line MDA-MB-231 overexpressing NRP-1. Delivery of reporter genes expressing either β-galactosidase (β-gal) or green fluorescent protein (GFP) was selectively enhanced in these cells when compared with NRP-1-negative cells. In MDA-MB-231 cells, an increase by 250% in β-gal activity was observed when delivered by lipopeptide-based liposomes compared to cationic liposomes alone.

We have synthesised a novel cholesterol-based cationic lipid to promote DNA transfer in cells. This lipid, dimethyl hydroxyethyl aminopropane carbamoyl cholesterol iodide (DMHAPC-Chol) contains a biodegradable carbamoyl linker and a hydroxyethyl group in the polar amino head moiety and is characterised by NMR. Liposomes prepared from this lipid and dioleoyl phosphatidyl ethanolamine (DOPE) in equimolar proportion showed a weak cytotoxicity as revealed by MTT assays and are efficient to deliver plasmids DNA evaluated by the expression of reporter genes in vitro and in vivo. In this paper, we present an original method to determine the lipid concentration based on the colorimetric detection of the colipid DOPE and the measure of the molar ratio DOPE/cationic lipid in the liposome by FTIR spectroscopy. The liposomes and lipid/DNA complexes structures were characterized by transmission electron microscopy (TEM) and by quasi-elastic light scattering (QLS). TEM indicated that the complexes correspond to aggregates containing globular substructures with liposomes size. The method of immuno-gold labelling was used to detect plasmid in the complex and reveals the presence of DNA inside the aggregates. Transfection results showed efficient DNA transfer depending on the charge ratio and liposomes conditioning. Gel retardation results indicated that at a molar charge ratio between X = 1.5 and X = 2.5 (depending on the liposome conditioning), all DNA was taken by liposomes. We showed that conditioning by freeze-drying (lyophilization) facilitates storage and improves transfection efficiency. When the liposomes were lyophilized prior to DNA addition or when the complexes were subjected to freeze-thawing cycles, the obtained complexes showed a transfection with levels enhanced up to four and five-fold respectively for the lyophilized liposomes and freeze-thawed complexes. NMR was used to characterize the modifications under freezing which showed an effect on 31P spectra.

A series of four cationic lipids derived from cholesterol was synthesised and their efficiencies to vectorise nucleic acids were compared. The investigation concerns the effects of systematic chemical modifications in the polar head and in the spacer. The cationic lipid molecules used are in the same family of 3beta[N-(N',N',N'-trimethylaminoethane)-carbamoyl] cholesterol iodide (TMAEC-Chol), presenting a spacer of two or three carbons and a quaternary ammonium polar head ramified with methyl or ethyl groups. These lipids formed stable liposomes sizing from 100 to 200 nm when prepared with the colipid dioleoyl phosphatidylethanolamine (DOPE). The goal of this work was to investigate the effect of the chemical structure of these cationic lipids on lipofection. Their ability to form complexes with DNA, their cytotoxicity and their transfection efficiency in vitro and in vivo were studied. Results were compared with those obtained from the well known cholesterol-based cationic lipid DC-Chol.In a melanoma cell line (B16-F10), results showed that either the polar head or the spacer affected the cytotoxicity. Cationic lipids with three ethyl groups in the head are more toxic than those with three methyl groups while cationic lipids with three carbons in the spacer are less toxic than those with two carbons in the spacer. The best transfection level was obtained in vitro and in vivo with cationic lipids having 3C in the spacer. Data indicated that among these lipids, in vivo gene transfer is advantaged by the methylated polar head while in vitro the best level was obtained with the ethylated one.Finally, it was observed that the chemical structure influences the transfection in the presence of serum while the complex charge and the DOPE ratios in liposomes preferentially affect the interaction with erythrocytes. Argumentations are proposed to explain the discrepancies between in vitro and in vivo transfection results concerning the optimal charge ratio and the chemical nature of the cationic lipid head group.

The determination of chemiluminescent intensity of reporter gene expression in vivo is generally disturbed by the presence of hemoglobin. Current methods consist in using perfusion to eliminate blood from investigated tumors or organs. In this work we propose a simple method to overcome this difficulty. The method consists in establishing an absorbance-dependence plot of the ratio R% = phi/phi(0) between the chemiluminescent intensities measured when hemoglobin is present or absent. For every measurement of the luminescent intensity phi on sample containing blood, if the absorbance A of the hemoglobin is determined, it allows one to have the intensity ratio R% which in turn gives the corrected intensity phi(0) when the absorption by hemoglobin is eliminated. The method is particularly adapted for comparative measurements of transfection levels in tumors where perfusion cannot be easily performed.

We investigated by transmission electron microscopy the cellular route in tumor MCF7 cells of DNA labeled with digoxigenin, carried by cationic liposomes (Lip+) prepared from TMAEC-Chol [3 beta(N-(N',N',N'-trimethylaminoethane)-carbamoyl)cholesterol iodide] and TEAPC-Chol [3 beta(N-(N',N',N'-triethylaminopropane)-carbamoyl)cholesterol iodide], two cholesterol-based cationic lipids containing a quaternary ammonium. In a previous work we showed the pathway of cationic lipid/plasmid complexes from the beginning of endocytosis until their entry into the perinuclear area. Beyond this limit, unlabeled exogenous plasmids cannot be distinguished with nuclear DNA. This work dealt with the cellular fate of cationic liposome-vectorized plasmids labeled with digoxigenin using an immunogold procedure. Early after the beginning of transfection (30 min, 1 hr, 5 hr), gold particles were observed only in the cytoplasm and in endosome-like vesicles, whereas after 24 hr gold particles were densely present in the nucleus. These results demonstrate the nuclear localization of plasmids vectorized by the cationic liposomes used. The results are discussed in comparison with transfection efficiency measurements.

This paper reports results concerning the transfection of gliosarcoma cells 9L using an original cholesterol-based cationic liposome as carrier. This cationic liposome was prepared from triethyl aminopropane carbamoyl cholesterol (TEAPC-Chol) and a helper lipid, dioleoyl phosphatidyl ethanolamine (DOPE). The used concentration of liposome was not cytotoxic as revealed by the MTT test. TEAPC-Chol/DOPE liposomes allowed the plasmids encoding reporter genes to enter the nucleus as observed both by electron microscopy and functionality tests using fluorescence detection of green fluorescent protein (GFP) and luminometric measurements of luciferase activity. By changing the cationic lipid/DNA molar charge ratio, optimal conditions were determined. Further, improvement of the transfection level has been obtained by either precondensing plasmid DNA with poly-l-lysine or by adding polyethylene glycol (PEG) in the transfection medium. The optimal conditions determined are different depending on whether the transfection is made with cells in culture or with tumors induced by subcutaneous (s.c.) injection of cells in Nude mice. For in vivo assays, a simple method to overcome the interference of haemoglobin with the chemiluminescence intensity of luciferase has been used. These results would be useful for gaining knowledge about the potential for the cationic liposome TEAPC-Chol/DOPE to transfect brain tumors efficiently.

The deviation in excess thermodynamic parameters such as molar volume (VE ), viscosity ( ηE), dielectric constant (ϵE ), Gibbs energy of activation of the viscous flow (G*E ) and surface tension ( γE) have been determined for propylene carbonate and 2,2,2-trifluoroethyl methyl carbonate (TFMC) mixtures atT= 298.15 K and for ethylene carbonate and TEMC mixtures at T= 313.15 K. All quantities were plotted against mole fraction over the whole concentration range. Polynomial regressions have been fitted with the results. The strength and the nature of the interactions between like and unlike components have been discussed.

The micellization and adsorption of two short chain perfluorodiols 3,3,4,4,5,5,6,6,6-nonafluorohexane-1,2-diol (nFHD) and 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctane-1,2-diol (tFOD) are examined from a thermodynamic point of view as a function of temperature and methanol content. The microenvironment of the fluorinated aggregates is evaluated by the fluorescence probe method using pyrene and a molecular rotor 1,1-dicyano-4-p-dimethylaminophenyl)-1,3-butadiene (DMAPhC). The formation of micellar aggregates being evidenced, the results are discussed in terms of the polarity and of the cohesion behavior of the micellar aggregates by taking into account the methanol (MeOH) effect. The critical micellization concentrations thus determined are compared with those given by surface tension measurements. Micellar and adsorption thermodynamic parameters such as Gibbs free energies, enthalpies, and entropies are determined together with the surface areas. The results are compared with literature data and discussed. A model for describing the adsorption process of the fluorinated compounds upon the influence of methanol is finally proposed. Copyright 2001 Academic Press.

The progress of research in gene therapy allows hope for treatment of mitochondrial genetic disorders provided that efficient methods for gene transfer into mitochondria can be found. In this work, we have used an oligonucleotide coupled covalently to a mitochondria-targeted peptide at one end and a cationic liposome prepared from trimethyl aminoethane carbamoyl cholesterol iodide (TMAEC-Chol) to carry it in living cells. With a fluorescent probe to label the oligonucleotide at the other end and by means of confocal microscopy, we show that such modified oligonucleotides complexed to liposomes enter into the cytoplasm of human fibroblasts in primary culture, and then, after dissociation from the complexes, they penetrate into the mitochondria. The fluorescence was still observed after 8 days, suggesting the continued presence of oligonucleotides. At the concentrations used for this study, the cationic liposomes have practically no effect on cell growth, as revealed by the MTT assay.

We have investigated the delivery and the pathway in tumoral MCF7 cells of DNA carried by liposomes prepared from (trimethyl aminoethane carbamoyl cholesterol iodide (TMAE-Chol), a cholesterol-based cationic lipid with a quaternary ammonium on the polar head. The structure of DNA-liposome complexes depends on the length of DNA and on the lipid-DNA charge ratio X. Spherical beads constitute fine structures of the observed complexes even when they appear as aggregates. For oligonucleotide transfer, dissociation from liposomes after transfection, penetration of the oligonucleotides into nuclei, and a long resident time were observed. For plasmid transfer, a correlation between the variation in the transfection level and the ultrastructure of complexes was demonstrated. The results showed a cellular route of lipid/plasmid complexes from the beginning by endocytosis, entrapped into endosomes, released by the latter until entry in the perinuclear area, and then penetration of plasmids inside the nuclei resulting in the observed expression of the beta-galactosidase gene.

The micellar and surface-active properties of two short-chain, non-ionic perfluorinated surfactants: 3,3,4,4,5,5,6,6,6-nonafluorohexane-1,2-diol (NFHD) and 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctane-1,2-diol (TFOD), have been investigated by the surface tension method. The influence of temperature and of methyl alcohol content on their aqueous solution behaviour has been studied. Both compounds present a sharp increase in the critical micellar concentration (CMC) as the methyl alcohol content increases. The concentrations of this solvent required for the disappearance of micelles (CDM), are 25 and 50% (in volume) for NFHD and TFOD, respectively. Their aqueous solutions have extremely low surface tensions, ∼15 mN m−1 over the CMC. Temperature has a weak effect on the solution behaviour of NFHD, whereas the CMC of TFOD increases significantly with temperature.