1-2-dielaidoylphosphatidylethanolamine and dioctadecylamidoglycylspermine

A monocationic lipid, YKS-220, with a symmetrical and biodegradable structure can be used as an effective gene transfer vector in a cationic particle form (not a cationic liposome form), and is obtained by diluting an ethanol solution of YKS-220 and DOPE (1:5, molar ratio) with an aqueous medium. This preparation method is more convenient than that for cationic liposomes. YKS-220 cationic particles showed a heterogeneous large mean diameter of 4.4 microm. An obvious size change was not observed when plasmid DNA was added. The transfection activity of YKS-220 cationic particles was comparable to those of YKS-220 liposomes and DOSPA liposomes (LipofectAMINE), and even higher than that of DOGS (TRNSFECTAM). Interestingly, the YKS-220 cationic particle/DNA complexes were resistant to the neutralizing effect of serum. All of these findings indicate that YKS-220 cationic particles are a convenient and efficient gene delivery reagent.

Topics: Animals; Cation Exchange Resins; Cations; Cattle; Cell Line; Cricetinae; Culture Media; Evaluation Studies as Topic; Fatty Acids, Monounsaturated; Gene Expression; Gene Transfer Techniques; Glycine; Humans; Indicators and Reagents; Lipids; Luciferases; Particle Size; Phosphatidylethanolamines; Plasmids; Quaternary Ammonium Compounds; Spermine; Transfection

We have characterized a new synthetic gene delivery system, termed DLS, which may be suitable for systemic gene therapy. DLS constitutes a lipopolyamine and a neutral lipid and associated plasmid DNA in the formation of lamellar vesicles (DLS-DNA). The ratio of lipids and lipid to DNA as well as the method of preparation were optimized to yield a high in vitro transfection efficiency compared with that previously reported for cationic lipid systems. DLS-DNA showed a rapid cellular uptake and distribution in the cytoplasmic and nuclear (especially in the nucleoli) compartments as determined by laser-assisted confocal microscopy. There was little or no plasmid DNA degradation over a period of 20 min, relatively slow plasma clearance, and effective and rapid cellular uptake of DLS-DNA following intravenous administration in mice. Supercoiled plasmid DNA could be detected in blood cells up to 1 h after injection. Systemic administration of DLS-DNA yielded transgene expression in mouse tissues, such as in lung or liver. The ratio of DLS:DNA and the procedure used to form DLS-DNA affected both the level and cellular specificity of expression of a luciferase reporter gene showing that in vitro transfection efficiency of DLS-DNA formulations cannot be easily extrapolated to an in vivo setting. Optimization of the formulation of a DNA delivery system was critical to obtain a defined structure resulting in a preparation with high reproducibility and stability, greater homogeneity of particle size and high efficacy following systemic gene transfer. In addition, the DLS system may be formulated for specific target tissues and may have a wide range of applications for gene therapy.

Topics: Animals; beta-Galactosidase; DNA; Gene Expression; Genetic Therapy; Genetic Vectors; Glycine; HeLa Cells; Humans; In Situ Hybridization; Liposomes; Luciferases; Mice; Mice, Inbred BALB C; Microscopy, Confocal; Microscopy, Electron; Phosphatidylethanolamines; Plasmids; Spermine; Transfection; Transgenes

Cationic liposomes have been proposed as alternative to adenovirus in the treatment of cystic fibrosis lung disease. Therefore, we have investigated the efficiency of two lipid mixtures in mediating gene transfer in in vitro and in vivo models. The cationic lipid DOTMA (N-(1-(2,3(dioleyloxy)propyl)-n,n,n-trimethylammoniumchloride++ +) and DOGS (dioctadecylamidoglycylspermine) were used in combination with the neutral lipid DOPE (dioleoylphosphatidylethanolamine). The relative transfection efficiencies of the two cationic liposomes were tested using the bacterial beta-galactosidase (lacZ) and the firefly luciferase genes. Gene expression was detected in both cell limes and primary culture of rhesus monkey airway epithelium after transfection with plasmid DNA complexed with DOGS/DOPE or DOTMA/DOPE. Transfection efficiency of both types of lipids was higher in the mouse fibroblast 3T3 cell line as compared to human carcinoma A549 cells and primary epithelial cultures. Administration of DNA-liposome complexes via intratracheal instillation resulted in expression of the lacZ and luciferase marker gene in the mouse airways. In vivo transfection mediated by both types of liposomes were proven to be far less efficient than adenovirus treatment.

Topics: 3T3 Cells; Adenoviridae; Animals; beta-Galactosidase; Bronchi; Cations; Cell Line; Cells, Cultured; Cystic Fibrosis; DNA, Recombinant; Drug Carriers; Female; Gene Expression; Genes, Reporter; Genetic Vectors; Glycine; Humans; Instillation, Drug; Liposomes; Luciferases; Lung; Macaca mulatta; Mice; Mice, Inbred BALB C; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Recombinant Fusion Proteins; Spermine; Trachea; Transfection

We have examined the complement-activating properties of synthetic cationic molecules and their complexes with DNA. Commonly used gene delivery vehicles include complexes of DNA with polylysine of various chain lengths, transferrin-polylysine, a fifth-generation poly(amidoamine) (PAMAM) dendrimer, poly(ethyleneimine), and several cationic lipids (DOTAP, DC-Chol/DOPE, DOGS/DOPE, and DOTMA/DOPE). These agents activate the complement system to varying extents. Strong complement activation is seen with long-chain polylysines, the dendrimer, poly(ethyleneimine), and DOGS (half-maximal at about 3 microM amine content in the assay used). Compared to these compounds, the other cationic lipids (in liposome formulations) are weak activators of the complement system (half-maximal approximately 50-100 microM positive charge in assay). Complement activation by polylysine is strongly dependent on the chain length. Short-chain oligolysines are comparable to cationic lipids in their activation of complement. Incubation of these compounds with DNA to form complexes reduces complement activation in virtually all cases. The degree of complement activation by DNA complexes is strongly dependent on the ratio of polycation and DNA (expressed as the charge ratio) for polylysine, dendrimer, poly(ethyleneimine), and DOGS. To a lesser degree, charge ratio also influences complement activation by monovalent cationic lipid-DNA complexes. For polylysine-DNA complexes, complement activation can be considerably reduced by modifying the surface of preformed DNA complexes with polyethyleneglycol (half-maximal approximately 20 microM amine content). The data suggests that, by appropriate formulation of DNA complexes, complement activation can be minimized or even avoided. These findings should facilitate the search for DNA complex formulations appropriate for reproducible intravenous gene delivery.

Topics: Animals; Cations; Complement Activation; DNA, Recombinant; Fatty Acids, Monounsaturated; Gene Transfer Techniques; Genetic Vectors; Glycine; Humans; Injections, Intravenous; Liposomes; Phosphatidylethanolamines; Phospholipids; Polylysine; Quaternary Ammonium Compounds; Sheep; Spermine

Direct in vivo transfection of tumor nodules in situ via liposome-DNA complexes has been employed as a strategy to accomplish antitumor immunization. To circumvent the potential safety hazards associated with systemic localization of delivered DNA, the utility of mRNA transcript-mediated gene delivery was explored. Capped, polyadenylated mRNA transcripts encoding the firefly luciferase and Escherichia coli lacZ reporter genes were derived by in vitro transcription. Transfection of the human breast cancer cell line MDA-MB-435 in vitro was accomplished employing cationic liposome-mRNA complexes. Evaluation of a panel of cationic liposome preparations demonstrated significant differences in the capacity of the various preparations to accomplish mRNA-mediated transfection. Quantitative evaluation of in vitro transfection demonstrated that target cells could be transfected at a high level of efficiency. The mRNA liposome-complexes were evaluated for in vivo transfection of tumor nodules in human xenografts in athymic nude mice. It could be demonstrated the liposome-mRNA complexes were comparable in efficacy to liposome-DNA complexes in accomplishing in situ tumor transfection. Thus, mRNA may be considered as an alternative to plasmid DNA as a gene transfer vector for genetic immunopotentiation applications.

Topics: beta-Galactosidase; Breast Neoplasms; Cation Exchange Resins; Cations; Drug Carriers; Fatty Acids, Monounsaturated; Genes, Reporter; Genetic Therapy; Glycine; Humans; Lipids; Liposomes; Luciferases; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Recombinant Fusion Proteins; RNA, Messenger; Safety; Spermine; Transfection; Tumor Cells, Cultured