RESUMEN
BACKGROUND: Colloidal stability of lipid/DNA aggregates is a major requirement for cationic lipid-mediated transfection which is particularly difficult to fulfil at the high DNA concentrations used for in vivo gene delivery. Thus, we have investigated the potential of poly(ethyleneglycol) (PEG) conjugates for steric stabilization of lipoplexes formed by bis(guanidinium)-tren-cholesterol/dioleoyl phosphatidylethanolamine (BGTC/DOPE) liposomes, a class of cationic liposomes we have developed over the past few years. METHODS AND RESULTS: We demonstrate that adequate lipophilic PEG derivatives can stabilize BGTC/DOPE lipoplexes formed at high DNA concentration. We also report the results of cryotransmission electron microscopy studies indicating that PEG-stabilized lipoplexes form DNA-coated structures which assemble into clusters exhibiting various complex morphologies. Finally, we report data from in vivo transfection experiments suggesting that PEG-mediated colloidal stabilization of concentrated lipoplex solutions may allow enhanced transfection of the mouse airways via intranasal administration. CONCLUSION: Our results represent an important step towards the design of multimodular BGTC-based systems for improved in vivo gene transfection.
Asunto(s)
Cloranfenicol/análogos & derivados , Colesterol/análogos & derivados , Colesterol/genética , Glicerofosfolípidos/genética , Pulmón/metabolismo , Fosfatidiletanolaminas , Transfección , Animales , Supervivencia Celular , Cloranfenicol/metabolismo , Colesterol/química , Colesterol/metabolismo , ADN/química , ADN/ultraestructura , Femenino , Técnicas de Transferencia de Gen , Vectores Genéticos , Glicerofosfolípidos/química , Glicerofosfolípidos/metabolismo , Guanidinas/química , Guanidinas/metabolismo , Ratones , Ratones Endogámicos BALB C , Polietilenglicoles/química , Células Tumorales CultivadasRESUMEN
One of the main challenges of gene therapy remains the increase of gene delivery into eukaryotic cells. We tested whether intracellular DNA release, an essential step for gene transfer, could be facilitated by using reducible cationic DNA-delivery vectors. For this purpose, plasmid DNA was complexed with cationic lipids bearing a disulphide bond. This reduction-sensitive linker is expected to be reduced and cleaved in the reducing milieu of the cytoplasm, thus potentially improving DNA release and consequently transfection. The DNA--disulphide-lipid complexation was monitored by ethidium bromide exclusion, and the size of complexes was determined by dynamic light scattering. It was found that the reduction kinetics of disulphide groups in DNA--lipid complexes depended on the position of the disulphide linker within the lipid molecule. Furthermore, the internal structure of DNA--lipid particles was examined by small-angle X-ray scattering before and after lipid reduction. DNA release from lipid complexes was observed after the reduction of disulphide bonds of several lipids. Cell-transfection experiments suggested that complexes formed with selected reducible lipids resulted in up to 1000-fold higher reporter-gene activity, when compared with their analogues without disulphide bonds. In conclusion, reduction-sensitive groups introduced into cationic lipid backbones potentially allow enhanced DNA release from DNA--lipid complexes after intracellular reduction and represent a tool for improved vectorization.
Asunto(s)
ADN/metabolismo , Técnicas de Transferencia de Gen , Metabolismo de los Lípidos , Cationes , Cromatografía Líquida de Alta Presión , Células HeLa , HumanosRESUMEN
[see structure]. We report the synthesis of new cationic lipids. These amphiphiles present a hydrophobic domain connected to a guanidinium entity by an unsaturated glycoside scaffold. The synthetic strategy using amide or acetal linkage led to various mono- and bicatenar derivatives. Investigation of their physicochemical properties indicated that these new compounds compact DNA.
Asunto(s)
Glicósidos/química , Lípidos/síntesis química , Cationes , ADN/química , Conformación MolecularRESUMEN
We have designed and synthesized original cationic lipids for modulated release of DNA from cationic lipid/DNA complexes. Our rationale was that modulated degradation of the lipids during or after penetration into the cell could improve the trafficking of DNA to the nucleus resulting in increased transgene expression. The new reduction-sensitive lipopolyamines (RSL) harbor a disulfide bridge within different positions in the backbone of the lipids as biosensitive function. A useful synthetic method was developed to obtain, with very good yields and reproducibility, unsymmetrical disulfide-bridged molecules, starting from symmetrical disulfides and thiols. The new lipopolyamines are good candidates as carriers of therapeutic genes for in vivo gene delivery. To optimize the transfection efficiency in these novel series, we have carried out structure-activity relationship studies by placing the disulfide bridge at different positions in the backbone of the cationic lipid and by systematic variation of lipid chain length. Results indicate that the transfection level can be modulated as a function of the location of the disulfide bridge in the molecule. We suggest that an early release of DNA during or after penetration into the cell, probably promoted by reduction of a disulfide bridge placed between the polyamine and the lipid, implies a total loss of transfection efficiency. On the other hand, proper modulation of DNA release by inserting the disulfide bridge between one lipid chain and the rest of the molecule brings about increased transfection efficiency as compared to previously described nondegradable lipopolyamine analogues. Finally, preliminary physicochemical characterization of the complexes demonstrates that DNA release from complexes can be modulated as a function of the surrounding reducing conditions of the complexes and of the localization of the disulfide bridge within the lipopolyamine. Our results suggest that RSL is a promising new approach for gene delivery.
Asunto(s)
ADN/genética , Técnicas de Transferencia de Gen , Lípidos/síntesis química , Poliaminas/síntesis química , Transgenes , Animales , Proteínas Sanguíneas/farmacología , Bovinos , Línea Celular , ADN/química , Disulfuros/química , Fluorescencia , Expresión Génica , Humanos , Luz , Lípidos/química , Luciferasas/genética , Luciferasas/metabolismo , Oxidación-Reducción , Tamaño de la Partícula , Poliaminas/química , Dispersión de Radiación , Relación Estructura-Actividad , TransfecciónRESUMEN
The coupling of bacterial surface (S)-layer proteins to lipid membranes is studied in molecular detail for proteins from Bacillus sphaericus CCM2177 and B. coagulans E38-66 recrystallized at dipalmitoylphosphatidylethanolamine (DPPE) monolayers on aqueous buffer. A comparison of the monolayer structure before and after protein recrystallization shows minimal reorganization of the lipid chains. By contrast, the lipid headgroups show major rearrangements. For the B. sphaericus CCM2177 protein underneath DPPE monolayers, x-ray reflectivity data suggest that amino acid side chains intercalate the lipid headgroups at least to the phosphate moieties, and probably further beyond. The number of electrons in the headgroup region increases by more than four per lipid. Analysis of the changes of the deduced electron density profiles in terms of a molecular interpretation shows that the phosphatidylethanolamine headgroups must reorient toward the surface normal to accommodate such changes. In terms of the protein structure (which is as yet unknown in three dimensions), the electron density profile reveals a thickness lz approximately 90 A of the recrystallized S-layer and shows water-filled cavities near its center. The protein volume fraction reaches maxima of >60% in two horizontal sections of the S-layer, close to the lipid monolayer and close to the free subphase. In between it drops to approximately 20%. Four S-layer protein monomers are located within the unit cell of a square lattice with a spacing of approximately 131 A.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Lípidos de la Membrana/química , Aminoácidos/análisis , Bacillus/química , Fenómenos Biofísicos , Biofisica , Cristalografía por Rayos X , Fosfatidiletanolaminas/química , Análisis Espectral , Electricidad Estática , Rayos XRESUMEN
This work describes composite structures composed of lipid bilayer or tetraetherlipid monolayer films attached to solid supports with associated crystalline bacterial cell surface layers (S-layers). The bilayer system was established by making use of the strong chemisorption of a first monolayer of thiolipids (1-octadecanethiol or 1,2-dimyristoyl-sn-glycero-3-phosphothioethanol) on gold and attaching a second monolayer of 1,2-dipalmitoyl-sn-3-phosphatidylethanolamine by the Langmuir Schaefer technique. The tetraetherlipid monolayer was composed of Glycerol-dialkyl-nonitol tetraetherlipid (GDNT). The monolayer of GDNT exhibits the thickness of a bilayer with hydrophilic headgroups on both sides and a hydrophobic inner part. Isolated S-layer protein from Bacillus sphaericus (CCM2177, which was injected into the subphase of an LB-trough, recrystallized into a coherent monolayer at the solid supported phospholipid bilayer and at the tetraehtherlipid monolayer. The composite lipid/S-layer structures were stable enough to allow lifting from the air-water interface, rinsing in water, and transfer into a scanning force microscope.