ABSTRACT
The synthesis of cationic lipo-thiophosphoramidates, a new family of cationic lipids designed for gene delivery, is reported herein. This new class of lipids is less polar than its oxygenated equivalent the lipo-phosphoramidates. Fluorescence anisotropy and FRET were used to determine the fluidity and fusogenicity of the lipo-phosphoramidates 3a-b and lipo-thiophosphoramidates 7a-b. The determination of both the size and the zeta potential of the nano-objects (liposomes and lipoplexes) and the determination of the DNA binding ability of the liposomes have completed the physico-chemical characterizations of the cationic lipids studied. Finally, the cationic lipids 3a-b and 7a-c have been evaluated as synthetic vectors for gene transfection into a variety of mammalian cell lines. The lipo-thiophosphoramidate 7a proved to be an efficient and low toxicity synthetic vector even when used at low lipid to DNA charge ratios.
Subject(s)
Amides/chemistry , Chemical Phenomena , Thiotepa/chemistry , Transgenes , Amides/pharmacology , Anisotropy , Cations/chemistry , Cell Line , Cell Survival/drug effects , Humans , Liposomes/chemistry , Membranes, Artificial , Molecular Structure , Thiotepa/pharmacologyABSTRACT
Cationic lipophosphoramidates constitute a class of cationic lipids we have previously reported to be efficient for gene transfection. Here, we synthesized and studied a novel lipophosphoramidate derivative characterized by an arsonium headgroup linked, via a phosphoramidate linker, to an unconventional lipidic moiety consisting of two diunsaturated linoleic chains. Physicochemical studies allowed us to comparatively evaluate the specific fluidity and fusogenicity properties of the liposomes formed. Although corresponding lipoplexes exhibited significant but relatively modest in vitro transfection efficiencies, they showed a remarkably efficient and reproducible ability to transfect mouse lung, with in vivo transfection levels higher than those observed with a monounsaturated analogue previously described. Thus, these results demonstrate that this diunsaturated cationic lipophosphoramidate constitutes an efficient and versatile nonviral vector for gene transfection. They also invite further evaluations of the transfection activity, especially in vivo, of gene delivery systems incorporating the lipid reported herein and/or other lipids bearing polyunsaturated chains.
Subject(s)
Amides/chemical synthesis , DNA/administration & dosage , Linoleic Acids/chemical synthesis , Liposomes/chemistry , Phosphoric Acids/chemical synthesis , Amides/chemistry , Amides/pharmacokinetics , Animals , Anisotropy , Arsenicals/chemistry , Cations , Cell Line , Colloids , DNA/chemistry , DNA/pharmacokinetics , Humans , Linoleic Acids/chemistry , Linoleic Acids/pharmacokinetics , Liposomes/pharmacokinetics , Luciferases/biosynthesis , Luciferases/genetics , Mice , Phosphoric Acids/chemistry , Phosphoric Acids/pharmacokinetics , Structure-Activity Relationship , Tissue Distribution , Transfection , TransgenesABSTRACT
We report here a study on the physicochemical properties of cationic phospholipids liposomes used for lipoplex formulation and DNA transfer. The original cationic phospholipids synthesized in our laboratory are first presented with the liposome formulation process. The second part deals with the liposomes fusogenic properties studied by fluorescence resonant energy transfer (FRET). The nature of the cationic polar head and the formulation with or without a neutral colipid have a great influence on the FRET signal. The third part reports the study of the viscosity of the liposome by fluorescence anisotropy measurements. It has been observed that the vectors having a saturated lipid chain exhibit a more pronounced anisotropy than those having unsaturated lipid chains. Finally, liposomes formed by a mixture of phospholipids and DC-Chol (a rigid lipid) leads to increase the anisotropy denoting a more rigid liposome.
Subject(s)
DNA/chemistry , Drug Carriers/chemistry , Fluorescence , Lipids/chemistry , Cations/chemistry , Chemistry, Physical , DNA/genetics , Fluorescence Polarization , Fluorescence Resonance Energy Transfer , Fluorescent Dyes/metabolism , Gene Transfer Techniques , Genetic Vectors , Lipids/genetics , Liposomes/chemistry , Molecular Structure , Phospholipids/chemistry , Phospholipids/genetics , Rhodamines/metabolism , Spectrometry, Fluorescence , ViscosityABSTRACT
Coal is currently becoming an increasingly interesting fossil energy resource and that is the reason why its maritime transport, and hence the risk of collier accidents, increase. In this work, the environmental impact of an accidental coal immersion at sea is studied: the physicochemical effects are estimated using innovative experimental setups--a laboratory seawater canal called "polludrome" is used to evaluate the behaviour of coal particles submitted to a seawater flow, and a specifically designed tub is used to study the physicochemical consequences induced when coal is introduced into continuously renewed seawater. When coal is introduced into seawater, the most easily visible consequences are physical: fine coal particles reduce the daylight penetration up to 100% and move along with the flow, and coal chunks accumulate on the floor. Chemical effects are also measured: humic matters are dissolved from coal into seawater (up to 2 mg L(-1)), but no release of polycyclic aromatic hydrocarbons is evidenced. Some inorganic compounds are dissolved, among which manganese, whose concentrations can reach 1 microg L(-1). Fortunately, the results show that the environmental impact of this type of accident would remain limited.
Subject(s)
Coal/analysis , Environmental Monitoring , Seawater/analysis , Water Pollutants, Chemical/analysis , Accidents, Occupational , Humic Substances/analysis , Light , Magnesium/analysis , Particle Size , Risk Assessment , Ships , Time Factors , Water MovementsABSTRACT
The DNA compacting properties of polyamines (especially spermine) are well-known, hence the use of spermine as the cationic part in several synthetic DNA carriers. Here, we describe the synthesis of modified spermines, with a "lipophosphoramidate" as the lipidic part, and their use for efficient in vitro transfection. Physicochemical measurements (particle size, zeta potentials, pKa determination) and gel retardation assays were also performed. Theoretical membrane-disrupting ability was established by FRET. Taken together, our results indicate that lipophosphoramidates constitute an interesting alternative to "classical" lipidic parts of cationic lipids used as DNA carriers.
Subject(s)
DNA/metabolism , Drug Carriers/chemical synthesis , Gene Transfer Techniques , Liposomes/chemistry , Nanostructures/chemistry , Phosphatidylethanolamines/chemistry , Spermine/chemistry , Cations , DNA/administration & dosage , DNA/genetics , Drug Carriers/pharmacology , Fluorescence Resonance Energy Transfer , Models, Chemical , Particle Size , Transfection/methodsABSTRACT
The absorption and fluorescence spectra of carbaryl (CB), carbofuran (CF) and carbendazim (MBC) have been studied. Fluorescence lifetime and fluorescence quantum yields are also reported as well as the influence of pH, solvent and presence of humic acids on fluorescence. The limit of detection (LD) of the three compounds has been measured by direct analysis by laser-induced fluorescence (LIF) using a pulsed YAG laser with an Optical Parametric Oscillator (OPO) as excitation source and an Intensified Charged Coupled Device (ICCD) camera for the fluorescence detection. Instrumental LD found for CB, for MBC and for CF are respectively 4, 50 and 1000 ng L(-1). In tap water, the LD obtained is 800 ng L(-1) for MBC and 20,000 ng L(-1) for CF. For CB, the use of a time shift between excitation and emission allows to reach a LD of 20 ng L(-1) in tap water.