lipofectamine and (3-dimyristyloxypropyl)(dimethyl)(hydroxyethyl)ammonium

The problem of assessing in vivo activity of gene delivery systems is complex. The reporter gene must be carefully chosen depending on the application. Plasmids with strong promoters, enhancers and other elements that optimize transcription and translation should be employed, such as the CMVint and pCIS-CAT constructs. Formulation aspects of cationic lipid-DNA complexes are being studied in several laboratories, and the physical properties and molecular organization of the complexes are being elucidated. Likewise, studies on the mechanism of DNA delivery with cationic lipids are accumulating which support the basic concept that the complexes fuse with biological membranes leading to the entry of intact DNA into the cytoplasm. Naked plasmid DNA administered by various routes is expressed at significant levels in vivo. This observation is not restricted to skeletal and heart muscle, but has been observed in lung, dermis, and in undefined tissues following intravenous administration. Most of the widely available cationic lipids, including Lipofectin, Lipofectamine and DC-cholesterol have a very poor ability to enhance DNA expression above the baseline naked DNA level, at least in lung. In this report we have revealed a novel cationic lipid, DLRIE, which can significantly enhance CAT expression in mouse lung by 25-fold above the naked DNA level. Other compounds are currently being evaluated which can enhance the naked DNA expression even higher. Plasmid vector improvements have led to further increase in in vivo lung expression, so that the net improvement is > 5,000-fold. Results of this nature are advancing the pharmaceutical gene therapy opportunities for synthetic cationic lipid based gene delivery systems.

Topics: Animals; beta-Galactosidase; Cation Exchange Resins; Cations; Chloramphenicol O-Acetyltransferase; DNA, Recombinant; Dodecanol; Drug Administration Routes; Drug Carriers; Genes, Reporter; Genetic Therapy; Genetic Vectors; Lipids; Liposomes; Luciferases; Macromolecular Substances; Mice; Mice, Inbred BALB C; Myristic Acids; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Recombinant Fusion Proteins; Transfection

To optimise the high efficiency, non-viral transfer of DNA to retinal pigment epithelial (RPE) cells in vitro.. A mammalian expression vector (pcDNA3.1) containing a firefly luciferase (luc) cDNA was used to transfect RPE cells using different chemical methods; calcium phosphate, DEAE-dextran and, liposomes-based transfection techniques. Transfection was optimised for both dose and time of exposure. The efficiency of gene transfer and cytotoxicity was measured 48 hours post-transfection using luciferase and MTT assays, respectively. The percentage of transfected cells (using optimal conditions) was determined with a construct expressing a jellyfish green fluorescent protein (GFP) using flow cytometery.. Calcium phosphate and DEAE-dextran techniques failed to transfect the vector and led to high cytotoxicity. Liposomes-based methods successfully transferred the vector to RPE cells, but the efficiency varied for different liposomes; Tfx-50 > Lipofectin > Lipofectamine > Cellfectin > DMRIE-C. No significant cytotoxicity was observed with any of the liposome treatments. Optimal transfection was achieved with Tfx-50 at a 3:1 ratio of DNA:liposome; between 12-15% of cells being transfected.. Efficient and non-toxic transfer of functional genes into primary RPE cells in vitro can be successfuly achieved by liposomes-based techniques. Tfx-50 appears to be a promising non-viral vector for RPE gene transfer.

Topics: Aged; Calcium Phosphates; Cation Exchange Resins; DEAE-Dextran; DNA; Dose-Response Relationship, Drug; Flow Cytometry; Gene Expression; Genetic Vectors; Green Fluorescent Proteins; Humans; Lipids; Liposomes; Luciferases; Luminescent Proteins; Middle Aged; Phosphatidylethanolamines; Pigment Epithelium of Eye; Quaternary Ammonium Compounds; Time Factors; Transfection

The family of cationic lipid transfection reagents described here demonstrates a modular design that offers potential for the ready synthesis of a wide variety of molecular variants. The key feature of these new molecules is the use of Tris as a linker for joining the hydrophobic domain to a cationic head group. The molecular design offers the opportunity to conveniently synthesise compounds differing in charge, the number and nature of hydrophobic groups in the hydrophobic domain and the characteristics of the spacer between the cationic and hydrophobic moieties. We show that prototype reagents of this design can deliver reporter genes into cultured cells with efficiencies rivaling those of established cationic lipid transfection reagents. A feature of these reagents is that they are not dependent on formulation with a neutral lipid for activity.

Topics: Animals; beta-Galactosidase; Cation Exchange Resins; Cell Survival; CHO Cells; Cricetinae; Cross-Linking Reagents; Drug Design; Escherichia coli; Genes, Reporter; Lipids; Liposomes; Plasmids; Quaternary Ammonium Compounds; Transfection; Tromethamine

It is well established that cationic liposomes facilitate the delivery of DNA and offer substantial advantages over viral-based delivery systems. However, these synthetic vectors readily aggregate in liquid formulations which in clinical trials requires preparation of lipid/DNA complexes at the bedside immediately before injection. This temporal requirement could be eliminated if complexes were formulated as stable preparations that could be shipped, stored, and administered as needed. To this end, our study investigates the stability of lipid/DNA complexes during physical stresses that might be encountered during shipping and storage, i.e., agitation and freeze-thawing. Our data show that agitation significantly reduces transfection rates in complexes prepared with three different commercially available lipid formulations. Additional experiments indicate that slow freezing is more damaging than rapid freezing, and that sucrose is able to preserve transfection and complex size during freeze-thawing. These results are consistent with previous reports and demonstrate that frozen formulations may be suitable for maintaining transfection rates of lipid/DNA complexes. Under certain conditions, we observe a reproducible 3-fold increase in transfection after freeze-thawing that is prevented by high concentrations of sucrose. Together, these data suggest that physical stresses can alter structural characteristics of lipid/DNA complexes that can markedly affect rates of DNA delivery.

Topics: Animals; Cation Exchange Resins; COS Cells; DNA; Drug Carriers; Drug Stability; Fatty Acids, Monounsaturated; Freezing; Lipids; Liposomes; Quaternary Ammonium Compounds; Transfection

Cationic liposomes may be valuable for the delivery of anti-sense oligonucleotides, ribozymes, and therapeutic genes into human immunodeficiency virus type 1 (HIV-1)-infected and uninfected cells. We evaluated the toxicity of three cationic liposomal preparations, Lipofectamine, Lipofectin, and 1, 2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DMRIE) reagent, to HIV-infected and uninfected cells. Monocyte/macrophages were infected with HIV-1BaL and treated with liposomes in medium containing 20% fetal bovine serum (FBS) for 4 h or 24 h at 37 degree C. Uninfected monocytic THP-1 cells and chronically infected THP-1/HIV-1IIIB cells were treated with phorbol 12-myristate 13-acetate (PMA) and exposed to liposomes in the presence of 10% FBS. Toxicity was evaluated by the Alamar Blue assay and viral p24 production. The toxic effect of cationic liposomes was very limited with uninfected cells, although concentrations of liposomes that were not toxic within a few days of treatment could cause toxicity at later times. In HIV-1BaL-infected macrophages, Lipofectamine (up to 8 microM) and Lipofectin (up to 40 microM) were not toxic after a 4-h treatment, while DMRIE reagent at 40 microM was toxic. While a 4-h treatment of THP-1/HIV-1IIIB cells with the cationic liposomes was not toxic, even up to 14 days post-treatment, all three cationic liposomes were toxic to cells at the highest concentration tested after a 24-h treatment. Similar results were obtained with the Alamar Blue assay, Trypan Blue exclusion and a method that enumerates nuclei. Infected cells with relatively high overall viability could be impaired in their ability to produce virions, indicating that virus production appears to be more sensitive to treatment with the cationic liposomes than cell viability. Our results indicate that HIV-infected cells are more susceptible than uninfected cells to killing by cationic liposomes. The molecular basis of this differential effect is unknown; it is proposed that alterations in cellular membranes during virus budding cause enhanced interactions between cationic liposomes and cellular membranes.

Topics: Animals; Cation Exchange Resins; Cattle; Cell Line; Cell Survival; Cells, Cultured; Culture Media; HIV Seronegativity; HIV-1; Humans; In Vitro Techniques; Lipids; Liposomes; Macrophages; Monocytes; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Tetradecanoylphorbol Acetate; Virion; Virus Replication