aLaboratoire de Chimie Structurale et Spectroscopie Biomoléculaire (CSSB), CNRS UMR 7033, UFR de Médecine, Université Paris 13, 74 rue Marcel Cachin, Bobigny Cedex F93017, FrancebLaboratoire d'Oncologie Cellulaire et Moléculaire, EA 2360, UFR de Médecine, Université Paris 13, 74 rue Marcel Cachin, Bobigny Cedex F93017, FrancecLaboratoire de Physicochimie des Interfaces et des Milieux Réactionnels (PIMIR), EA 2098, Faculté des Sciences, Parc de Grandmont, Tours 37200, FrancedLaboratoire de Biothérapies Bénéfices et Risques, EA 3410, UFR de Médecine, Université Paris 13, 74 rue Marcel Cachin, Bobigny Cedex F93017, France
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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 Symbol spectra.