1-2-dielaidoylphosphatidylethanolamine 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

Lipid-DNA complexes (lipoplexes) are widely used, since several years, as gene carriers. However, their transfection efficiency, both in vitro and in vivo, depends, in a rather complex way, on different interconnected parameters, ranging from the chemical composition of the lipid components to the size and size distribution of the complexes and, moreover, to the composition of the suspending medium. In this paper, we have investigated the behavior of nine different commercially available transfection agents (liposomal and non-liposomal) and their lipoplexes, at different molar charge ratios and in different experimental conditions. The size and the time stability of the resulting lipoplexes were investigated by means of dynamic light scattering methods and their toxicity and transfection efficiency were assayed in vitro in a model tumor cell line (C6 rat glioma cell line). An attempt to correlate the different parameters governing the complex phenomenology observed has been made. Whereas all the formulations investigated display a low toxicity, that increases with the increase of the lipid-DNA molar charge ratio, the transfection efficiency markedly depends, besides the molar charge ratio, on the lipid composition and on the lipoplex size, in a rather correlated way. The aim of this work is to present, in a wide scenario, an example of the inter-correlation among the different parameters that influence the transfection efficiency of lipoplexes and to suggest the role exerted by the average size of the resulting aggregates in their overall effectiveness as carriers in gene therapy.

Topics: Animals; Cations; Cell Death; Cell Line, Tumor; Cell Survival; Cholesterol; DNA; Lipids; Liposomes; Particle Size; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Rats; Serum; Time Factors; Transfection

The aim of this study was to optimize non-viral gene transfer conditions and investigate the effect of fibroblast growth factor-1 (FGF-1) gene transfer on human corneal endothelial cell (HCEC) proliferation. Five non-viral vectors (Lipofectin, DMRIE-C, DAC-30, Effectene, FuGene6) were used to transfect HCEC with plasmids coding for enhanced green fluorescent protein (EGFP) and FGF-1. Transfection efficiency and toxicity (n=6) were quantified and optimized using the EGFP construct by FACS-analysis. Using optimal conditions HCEC were transfected with the FGF-1 plasmid and cell proliferation as well as expression of FGF-1 were determined at days 4 and 7 by counting and western blotting, respectively. Lipofectin (17+/-2.02%) transfected HCEC more successfully than DMRIE-C (11+/-1.46%), Effectene (9+/-0.62%), FuGene (9+/-0.93%) and DAC-30 (7+/-0.59%). Toxicity of the lipids ranged from 2 to 4%. Optimal HCEC proliferation was achieved with DAC-30/FGF-1 (P<0.05), whereas all other vectors did not result in significantly increased cell proliferation. However, all of the transfected cells produced FGF-1 in different amounts as indicated by western blotting. Efficient and almost non-toxic transfer of the FGF-1 gene into HCEC can be successfully achieved by lipid-based techniques. Using optimal conditions significantly increased cell proliferation was independent on gene transfer efficiency. This may indicate that even a low transfection rate is sufficient to produce a concentration of FGF-1 that will have a stimulatory effect on HCECs.

Topics: Blotting, Western; Cell Count; Cell Division; Cells, Cultured; Cholesterol; Endothelial Cells; Endothelium, Corneal; Fibroblast Growth Factor 1; Flow Cytometry; Genetic Vectors; Humans; Lipids; Liposomes; Microscopy, Electron; Phosphatidylethanolamines; Quaternary Ammonium Compounds; 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

Topics: Amino Acids; Cholesterol; Fatty Acids, Monounsaturated; Genetic Vectors; HeLa Cells; Humans; Indicators and Reagents; K562 Cells; Lipids; Luciferases; Phosphatidylethanolamines; Plasmids; Quaternary Ammonium Compounds; Recombinant Proteins

A cavitary glioblastoma model was created by injection of RT-2 cells, which express endogenous wild type p53, into the peritoneal cavity of nude mice. This model developed multiple layers of tumor cells invading the peritoneal surface and was used to mimic the postoperative surgical cavity remaining after glioblastoma (GBM) excision in patients. Rhodamine labeled DMRIE/DOPE + DNA complexes were found to penetrate at least 20 tumor cell layers. Injection of p53 gene/liposome complexes into the intraperitoneal cavity after the tumor was established resulted in massive tumor necrosis. Prominent staining of human p53 protein using the DO-1 antibody was found in tumor cells near the necrotic lesions. Tumor explants expressed human p53 protein and showed a 54% growth reduction in an in vitro growth assay. Further, DMRIE/DOPE mediated p53 gene transfection significantly increased the mean survival time of tumor bearing mice compared to vector control. These results demonstrate the efficiency of using exogenous wild type p53 to suppress glioblastoma cell with endogenous wild type p53 in vivo through liposome mediated transfection method.

Topics: Animals; Brain Neoplasms; Drug Delivery Systems; Gene Transfer Techniques; Genetic Therapy; Glioblastoma; Humans; Lipids; Liposomes; Mice; Mice, Nude; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Rats; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The mucosal surfaces represent the primary site for transmission of several viruses including HIV. To prevent mucosal transmission and dissemination to the regional lymph nodes, an effective HIV vaccine may need to stimulate immune responses at the genital and rectal mucosa. Optimal induction of mucosal immunity in general requires targeting antigens to the specialized antigen presenting cells of mucosal associated lymphoid tissues. The nasal mucosa may provide a simple, non-invasive route to deliver DNA encoding the introduced gene to stimulate mucosal immunity. As a first step to evaluate the feasibility of this approach, we have investigated as a model system, systemic and mucosal immune responses elicited to firefly luciferase generated by DNA immunization. Incorporating DNA into liposomes with cationic lipids enhanced luciferase expression in nasal tissue, and was associated with induction of a humoral response in serum and vaginal fluids and also a proliferative and cytotoxic T lymphocyte response in the spleen and iliac lymph nodes draining the genital and rectal mucosa.

Topics: Administration, Intranasal; AIDS Vaccines; Animals; DNA, Viral; Enzyme-Linked Immunosorbent Assay; Female; HIV Antibodies; HIV-1; Immunity, Mucosal; Lipids; Liposomes; Mice; Phosphatidylethanolamines; Quaternary Ammonium Compounds; T-Lymphocytes; T-Lymphocytes, Cytotoxic; Vaccines, DNA

We used a 32P-labeled pCMV-CAT plasmid DNA to estimate the DNA uptake efficiency and unlabeled pCMV-CAT plasmid DNA to quantify the CAT activity after transfection of COS cells using each of the three following cationic compounds: [1] vectamidine (3-tetradecylamino-N-tert-butyl-N'-tetradecylpropionamidine, and previously described as diC14-amidine [1]), [2] lipofectin (a 1:1 mixture of N-(1-2,3-dioleyloxypropyl)-N,N,N-triethylammonium (DOTMA) and dioleylphosphatidylethanolamine (DOPE)), and [3] DMRIE-C (a 1:1 mixture of N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl) ammonium bromide (DMRIE) and cholesterol). Surprisingly, a high CAT activity was observed with vectamidine although the DNA uptake efficiency was lower as compared to lipofectin and DMRIE-C. Transmission electron microscopy (TEM) revealed endocytosis as the major pathway of DNA-cationic lipid complex entry into COS cells for the three cationic lipids. However, the endosomal membrane in contact with complexes containing vectamidine or DMRIE-C often exhibited a disrupted morphology. This disruption of endosomes was much less frequently observed with the DNA-lipofectin complexes. This comparison of the three compounds demonstrate that efficient transfection mediated by cationic lipids is not only correlated to their percentage of uptake but also to their ability to destabilize and escape from endosomes.

Topics: Amidines; Animals; Cell Membrane; Chloramphenicol O-Acetyltransferase; COS Cells; Cytomegalovirus; Endosomes; Lipids; Liposomes; Phosphatidylethanolamines; Plasmids; Quaternary Ammonium Compounds; Recombinant Fusion Proteins; Transfection

Cationic liposomes are used to deliver genes into cells in vitro and in vivo. The present study is aimed to characterize the electrostatic parameters of cationic, large unilamellar vesicles, 110 +/- 20 nm in size, composed of DOTAP/DOPE (mole ratio 1/1), DOTAP/DOPC (mole ratio 1/1), 100% DOTAP, DMRIE/DOPE 1/1, or DC-CHOL/DOPE (mole ratio 1/1). {. DOTAP, N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine; DMRIE, 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide; DC-CHOL, 3beta[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol}. The cationic liposomes had a large positive surface potential and a high pH at the liposomal surface in 20 mM Hepes buffer (pH 7.4) as monitored by the pH-sensitive fluorophore 4-heptadecyl-7-hydroxycoumarin. In contrast to DOTAP and DMRIE which were 100% charged, DC-CHOL in DC-CHOL/DOPE (1/1) liposomes was only about 50% charged in 20 mM Hepes buffer (pH 7.4). This might result in an easier dissociation of bilayers containing DC-CHOL from the plasmid DNA (which is necessary to enable transcription), in a decrease of the charge on the external surfaces of the liposomes or DNA-lipid complexes, and in an increase in release of the DNA-lipid complex into the cytosol from the endosomes. Other electrostatic characteristics found were that the primary amine group of DOPE in cationic liposomes dissociated at high (> 7.9) pHbulk and that a salt bridge was likely between the quaternary amine of DOTAP or DMRIE and the phosphate group of DOPE or DOPC, but not between the tertiary amine of DC-CHOL and the phosphate group of DOPE. The liposomes containing DOTAP were unstable upon dilution, probably due to the high critical aggregation concentration of DOTAP, 7 X 10(-5) M. This might also be a mechanism of the dissociation of bilayers containing DOTAP from the plasmid DNA.

Topics: Cholesterol; Chromatography, High Pressure Liquid; Fatty Acids, Monounsaturated; Fluorescent Dyes; Gene Transfer Techniques; Hydrogen-Ion Concentration; Kinetics; Lipids; Liposomes; Models, Structural; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Static Electricity; Structure-Activity Relationship; Surface Properties; Umbelliferones

Cytofectins are positively charged lipophilic molecules that readily form complexes with DNA and other anionic polynucleotides. Normally, cytofectins are combined with an activity-augmenting phospholipid such as dioleoylphosphatidylethanolamine (DOPE), and a film of dried, mixed lipid is prepared and hydrated to form cationic liposomes. The liposome solution is then mixed with a plasmid DNA solution to afford cytofectin-DNA complexes which, when presented to living cells, are internalized and the transgene is expressed. One of the most potent cytofectins, dimyristoyl Rosenthal inhibitor ether (DMRIE), is presently being used to deliver transcriptionally active DNA into human tumor tissues. Here we report the remarkable consequences of replacing the alcohol moiety of DMRIE with a primary amine. The resulting cytofectin, called beta-aminoethyl-DMRIE (betaAE-DMRIE), promoted high level transfection over a broad range of DNA and cationic lipid concentrations. A comparison of in vitro transfection activity between DMRIE and betaAE-DMRIE in 10 cell types revealed that betaAE-DMRIE was more active than DMRIE, and that betaAE-DMRIE, unlike DMRIE, was maximally effective in the absence of colipid. The consequences of the alcohol-to-amine conversion on the structure of the cytofectin/DNA complex was also examined by Atomic Force Microscopy. Strikingly dissimilar images were found for plasmid DNA alone and for the plasmid complexes of betaAE-DMRIE and DMRIE/DOPE.

Topics: Alcohols; Amines; Animals; beta 2-Microglobulin; beta-Galactosidase; Cell Line; DNA; Drug Carriers; Genes, Bacterial; Genes, MHC Class I; HLA-B7 Antigen; Humans; Lipids; Liposomes; Phosphatidylethanolamines; Plasmids; Quaternary Ammonium Compounds; Recombinant Proteins; Structure-Activity Relationship; Transfection; Tumor Cells, Cultured

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

Using a minipump combined with stereotaxic techniques allows continuous delivery of therapeutic genetic materials into the brain. We investigated the therapeutic efficacy of liposome-mediated HSVtk gene transfer of experimental brain F98 glioma followed by treatment with ganciclovir. A single injection of DNA-liposome complexes showed a therapeutically significant decrease in the tumor volume. Continuous intracerebral delivery of DNA-liposome complexes using an osmotic minipump led to complete tumor regression in 36.4% of the treated animals. The safety and toxicity of this gene delivery system were also assessed. No organ pathology was observed in the experimental animals. The continuous gene delivery system could be a useful means of achieving higher doses with less toxicity and without the need for frequent injections.

Topics: Animals; Antiviral Agents; Brain Neoplasms; Cell Survival; DNA, Viral; Ganciclovir; Gene Transfer Techniques; Genes, Reporter; Genetic Therapy; Glioma; Humans; Lac Operon; Lipids; Liposomes; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Rats; Rats, Inbred F344; Simplexvirus; Stereotaxic Techniques; Thymidine Kinase

Cationic lipids are widely used for gene transfer in vitro and show promise as a vector for in vivo gene therapy applications. However, there is limited understanding of the cellular and molecular mechanisms involved. We investigated the individual steps in cationic lipid-mediated gene transfer to cultured cell lines. We used DMRIE/DOPE (a 1:1 mixture of N-[1-(2,3-dimyristyloxy) propyl]-N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide (DMRIE) and dioleoyl phosphatidylethanolamine (DOPE) as a model lipid because of its efficacy and because it is being used for clinical trials in humans. The data show that cationic lipid-mediated gene transfer is an inefficient process. Part of the inefficiency may result from the fact that the population of lipid-DNA complexes was very heterogeneous, even under conditions that have been optimized to produce the best transfection. Inefficiency was not due to inability of the complex to enter the cells because most cells took up the DNA. However, in contrast to previous speculation, the results indicate that endocytosis was the major mechanism of entry. After endocytosis, the lipid-DNA aggregated into large perinuclear complexes, which often showed a highly ordered tubular structure. Although much of the DNA remained aggregated in a vesicular compartment, there was at least a small amount of DNA in the cytoplasm of most cells. That observation plus results from direct injection of DNA and lipid-DNA into the nucleus and cytoplasm indicate that movement of DNA from the cytoplasm to the nucleus may be one of the most important limitations to successful gene transfer. Finally, before transcription can occur, the data show that lipid and DNA must dissociate. These results provide new insights into the physical limitations to cationic lipid-mediated gene transfer and suggest that attention to specific steps in the cellular process may further improve the efficiency of transfection and increase its use in a number of applications.

Topics: Cell Nucleus; DNA; Gene Transfer Techniques; HeLa Cells; Humans; Lipids; Myristic Acids; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Transcription, Genetic; Transfection

Cationic lipids offer several advantages for gene delivery, both in vitro and in vivo. However, high-efficiency gene transfer has been demonstrated only for limited cell types. Here, we examine the level of expression of a luciferase reporter gene, delivered using cationic lipids, in both cell lines and primary human cells including peripheral blood mononuclear cells and CD34(+)-enriched hematopoietic cells. Variables shown to affect the efficiency of gene expression included the type of lipid, the amounts of DNA and lipid, the day of assay following transfection, the media used for lipid:DNA complex formation, the cell number, the promoter driving expression of the reporter gene and the physiological state of the cells (e.g., whether or not cells were differentiated). The maximal luciferase expression observed with the primary cells was one to two orders of magnitude lower than that seen in cell lines. Further studies, possibly involving altering the growth conditions for the cells, or using episomal vectors that will allow extrachromosomal maintenance of the DNA, are required to improve the level of transgene expression in the primary human cell types used here.

Topics: Animals; Antigens, CD34; Cations; CD4-Positive T-Lymphocytes; Cell Line; DNA; Gene Expression; Gene Transfer Techniques; Genes, Reporter; Genetic Techniques; Genetic Vectors; Hematopoietic Stem Cells; Humans; Lipid Metabolism; Lipids; Luciferases; Lymphoma; Mice; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Spermine; Tumor Cells, Cultured

Among the potential nonviral vectors for human gene therapy are DNA-liposome complexes. In a recent clinical study, this delivery system has been utilized. In this report, a novel cationic lipid, dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium (DMRIE), has been substituted into the DNA-liposome complex with dioleoyl phosphatidylethanolamine (DOPE), which both improves transfection efficiencies and allows increased doses of DNA to be delivered in vivo. The safety and toxicity of this DNA-liposome complex has been evaluated in two species, mice and pigs. The efficacy of DMRIE/DOPE in inducing an antitumor response in mice after transfer of a foreign MHC has been confirmed. No abnormalities were detected after administration of up to 1,000-fold higher concentrations of DNA and lipid than could be tolerated in vivo previously. Examination of serum biochemical enzymes, pathologic examination of tissue, and analysis of cardiac function in mice and pigs revealed no toxicities related to this treatment. This improved cationic lipid formulation is well-tolerated in vivo and could therefore allow higher dose administration and potentially greater efficiency of gene transfer for gene therapy.

Topics: Animals; Cations; Female; Genetic Therapy; Genetic Vectors; Heart; Humans; Infusions, Intra-Arterial; Infusions, Intravenous; Lipids; Liposomes; Mice; Mice, Inbred BALB C; Myristic Acids; Organ Specificity; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Swine; Transfection