dioleoyl-phosphatidylethanolamine and dimethyldioctadecylammonium

Gene therapy is a potential method for treating a large range of diseases. Gene vectors are widely used in gene therapy for promoting the gene delivery efficiency to the target cells. Here, gold nanoparticles (AuNPs) coated with dimethyldioctadecylammonium bromide (DODAB)/dioleoylphosphatidylethanolamine (DOPE) are synthesized using a facile method for a new gene vector (DODAB/DOPE-AuNPs), which possess 3- and 1.5-fold higher transfection efficiency than those of DODAB-AuNPs and a commercial transfection agent, respectively. Meanwhile, it is nontoxic with concentrations required for effective gene delivery. Imaging and quantification studies of cellular uptake reveal that DOPE increases gene copies in cells, which may be attributed to the smaller size of AuNPs/DNA complexes. The dissociation efficiency of DNA from the endocytic pathway is quantified by incubating with different buffers and investigated directly in the cells. The results suggest that DOPE increases the internalization of AuNPs/DNA complexes and promotes DNA release from early endosomes for the vector is sensitive to the anionic lipid membrane and the decreasing pH along the endocytic pathway. The new vector contains the potential to be the new alternative as gene delivery vector for biomedical applications.

Topics: Animals; Anions; Buffers; Cell Count; Cell Death; DNA; Endocytosis; Female; Gene Transfer Techniques; Gold; HEK293 Cells; Humans; Hydrogen-Ion Concentration; Injections, Intramuscular; Lipids; Liposomes; Metal Nanoparticles; Mice, Inbred BALB C; Particle Size; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Static Electricity; Tissue Distribution; Transfection

Cationic liposome (CL)-DNA complexes (lipoplexes) have appeared as leading nonviral gene carriers in worldwide gene therapy clinical trials. Arriving at therapeutic dosages requires the full understanding of the mechanism of transfection. However, using CLs to deliver therapeutic nucleic acids and drugs to target organs have some problems, including low transfection efficiency. The aim of this study was developing novel CLs containing four neutral lipids; cholesterol, 1,2-dioleoyl phosphatidylethanolamine, distearoylphosphatidylcholine and dipalmitoylphosphatidylcholine as a helper lipid and dimethyl dioctadecyl ammonium bromide as a cationic lipid to increase transfection efficiency. We have investigated the correlation between number of lipid composition and transfection efficiency. The morphology, size and zeta potential of liposomes and lipoplexes were measured and lipoplexes formation was monitored by gel retardation assay. Transfection efficiency was assessed using firefly luciferase reporter assay. It was found that transfection efficiency markedly depended on liposome to plasmid DNA (pDNA) weight ratio, lipid composition and efficiency of pDNA entrapment. High transfection efficiency of plasmid by four component lipoplexes was achieved. Moreover, lipoplexes showed lower transfection efficiency and less cytotoxicity compared to Lipofectamine™. These results suggest that lipid composition of nanoliposomes is an important factor in control of their physical properties and also yield of transfection.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Cations; Cholesterol; DNA; Electrophoretic Mobility Shift Assay; HEK293 Cells; Humans; Liposomes; Nanoconjugates; Phosphatidylcholines; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Transfection

Successful drug delivery via lipid-based systems has often been aided by the incorporation of 'helper lipids'. While these neutral lipids enhance the effectiveness of cationic lipid-based delivery formulations, many questions remain about the nature of their beneficial effects. The structure of monolayers of the cationic lipid dimethyldioctadecylammonium bromide (DODAB) alone, and mixed with a neutral helper lipid, either diolelyphosphatidylethanolamine or cholesterol at a 1 : 1 molar ratio was investigated at the air-water interface using a combination of surface pressure-area isotherms, Brewster angle microscopy (BAM) and specular neutron reflectivity in combination with contrast variation. BAM studies showed that while pure DODAB and DODAB with cholesterol monolayers showed fairly homogeneous surfaces, except in the regions of phase transition, monolayers of DODAB with diolelyphosphatidylethanolamine were, in contrast, inhomogeneous exhibiting irregular bean-shaped domains throughout. Neutron reflectivity data showed that while the thickness of the DODAB monolayer increased from 17 to 24 Å as it was compressed from a surface pressure of 5-40 mN m(-1), the thickness of the helper lipid-containing monolayers, over the same range of surface pressures, was relatively invariant at between 25 and 27 Å. In addition, the monolayers containing diolelyphosphatidylethanolamine were found to be more heavily hydrated than the monolayers of cationic lipid, alone or in combination with cholesterol, with hydration levels of 18 molecules of water per molecule of lipid being recorded for the diolelyphosphatidylethanolamine-containing monolayers at a surface pressure of 30 mN m(-1) compared with only six and eight molecules of water per molecule of lipid for the pure DODAB monolayer and the cholesterol-containing DODAB monolayer, respectively.

Topics: Cholesterol; Drug Delivery Systems; Membrane Lipids; Microscopy; Neutrons; Phosphatidylethanolamines; Pressure; Quaternary Ammonium Compounds; Unilamellar Liposomes; Water

The interaction of DNA with monolayers of the cationic lipid dimethyldioctadecylammonium bromide, with/without 50 mol % of a neutral "helper" lipid, either dioleoylphosphatidylethanolamine or cholesterol, has been studied using specular neutron reflection, surface pressure-area isotherms, and Brewster angle microscopy. The amount of DNA bound to the lipid head groups has been comprehensively quantified in the range of 8-39 vol% of DNA with respect to the monolayer composition (monolayers composed of dimethyldioctadecylammonium bromide binding the most DNA and monolayers containing dioleoylphosphatidylethanolamine binding the least) and surface pressure (DNA binding being greatest at highest surface pressures). Surprisingly, regardless of these variables, the thickness of the DNA-containing layer remained approximately constant between 18 and 25 Å. This systematic study is the first direct quantification of the binding of DNA with two different helper-lipid-containing multicomponent monolayers, an important step toward understanding interaction parameters in more realistic models of gene delivery systems.

Topics: Cations; Cholesterol; DNA; Gene Transfer Techniques; Models, Chemical; Models, Molecular; Neutron Diffraction; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Surface Properties; Unilamellar Liposomes

Despite considerable interest and investigations on cationic lipid-DNA complexes, reports on lipid-RNA interaction are very limited. In contrast to lipid-DNA complexes where lipid binding induces partial B to A and B to C conformational changes, lipid-tRNA complexation preserves tRNA folded state. This study is the first attempt to investigate the binding of cationic lipid with transfer RNA and the effect of lipid complexation on tRNA aggregation and condensation. We examine the interaction of tRNA with cholesterol (Chol), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethylammoniumbromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE), at physiological condition, using constant tRNA concentration and various lipid contents. FTIR, UV-visible, CD spectroscopic methods and atomic force microscopy (AFM) were used to analyze lipid binding site, the binding constant and the effects of lipid interaction on tRNA stability, conformation and condensation. Structural analysis showed lipid-tRNA interactions with G-C and A-U base pairs as well as the backbone phosphate group with overall binding constants of K(Chol) = 5.94 (+/- 0.8) x 10(4) M(-1), K(DDAB) = 8.33 (+/- 0.90) x 10(5) M(-1), K(DOTAP) = 1.05 (+/- 0.30) x 10(5) M(-1) and K(DOPE) = 2.75 (+/- 0.50) x 10(4) M(-1). The order of stability of lipid-tRNA complexation is DDAB > DOTAP > Chol > DOPE. Hydrophobic interactions between lipid aliphatic tails and tRNA were observed. RNA remains in A-family structure, while biopolymer aggregation and condensation occurred at high lipid concentrations.

Topics: Cations; Cholesterol; Circular Dichroism; Fatty Acids, Monounsaturated; Hydrophobic and Hydrophilic Interactions; Lipids; Microscopy, Atomic Force; Nucleic Acid Conformation; Phosphates; Phosphatidylethanolamines; Quaternary Ammonium Compounds; RNA, Transfer; Spectroscopy, Fourier Transform Infrared

Adenoviral vectors have been commonly used in gene therapy protocols, however the success of their use is often limited by the induction of host immunity to the vector. Following exposure to the adenoviral vector, adenoviral-specific neutralising antibodies are produced which limits further administration. This study examines the efficacy of complexing liposomes to adenovirus for the protection of the adenovirus from neutralising antibodies in an in vitro setting. Dimethyldioctadecylammonium bromide (DDAB)-dioleoyl-l-phosphatidylethanolamine (DOPE) liposomes were bound at varying concentrations to adenovirus to form AL complexes and tested these complexes' ability to prevent adenoviral neutralisation. It is shown that by increasing the concentration of liposomes in the adenoviral-liposome (AL) complexes we can increase the level of immuno-shielding afforded the adenovirus. It is also shown that the increase in liposomal concentration may lead to drawbacks such as increased cytotoxicity and reductions in expression levels.

Topics: Adenoviridae; beta-Galactosidase; Cell Line; Gene Expression; Genes, Reporter; Genetic Vectors; Humans; Liposomes; Neutralization Tests; Phosphatidylethanolamines; Quaternary Ammonium Compounds

The transfer of plasmid expression vectors to cells is essential for transfection after administration of lipid-based DNA formulations (lipoplexes). A murine i.p. B16/BL6 tumor model was used to characterize DNA delivery, liposomal lipid delivery, and gene transfer after regional (i.p.) administration of free plasmid DNA and DNA lipoplexes. DNA lipoplexes were prepared using cationic dioleoyldimethylammonium chloride/dioleoylphosphatidylethanolamine (50:50 mol ratio) liposomes mixed with plasmid DNA (1 microgram DNA/10 nmol lipid). The plasmid used contained the chloramphenicol acetyltransferase gene and chloramphenicol acetyltransferase expression (mU/g tumor) was measured to estimate transfection efficiency. Tumor-associated DNA and liposomal lipid levels were measured to estimate the efficiency of lipid-mediated DNA delivery to tumors. Plasmid DNA delivery was estimated using [3H]-labeled plasmid as a tracer, dot blot analysis, and/or Southern analysis. Liposomal lipid delivery was estimated using [14C]-dioleoylphosphatidylethanolamine as a liposomal lipid marker. Gene expression in the B16/BL6 tumors was highly variable, with values ranging from greater than 2,000 mU/g tumor to less than 100 mU/g tumor. There was a tendency to observe enhanced transfection in small (<250 mg) tumors. Approximately 18% of the injected dose of DNA was associated with these small tumors 2 h after i.p. administration. Southern analysis of extracted tumor DNA indicated that plasmid DNA associated with tumors was intact 24 h after administration. DNA and associated liposomal lipid are efficiently bound to tumors after regional administration; however, it is unclear whether delivery is sufficient to abet internalization and appropriate subcellular localization of the expression vector.

Topics: Animals; Blotting, Southern; Chloramphenicol O-Acetyltransferase; Escherichia coli; Female; Gene Transfer Techniques; Genetic Vectors; Injections, Intraperitoneal; Lipids; Liposomes; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Phosphatidylethanolamines; Plasmids; Quaternary Ammonium Compounds; Tissue Distribution

Intratracheal (i.t.) and intravenous (i.v.) delivery of DNA-vector formulations are two strategies to obtain gene transfer to the lung, it is still uncertain, however, which of these two modes of delivery will be more effective in the treatment of cystic fibrosis and other lung diseases. In this study, we attempted to optimize formulations of the cationic liposome DODAC:DOPE (dioleoyldimethylammonium-chloride: dioleoylphosphatidylethanolamine) complexed to plasmids encoding chloramphenicol acetyltransferase for i.t. and i.v. injection into CD-2 mice and compared the two methods. Our results showed that both methods conferred reporter gene expression in the lung that was significantly higher relative to injection of plasmid DNA alone. Expression using either mode of administration was maximal 24 h after injection and declined to around 10% of day 1 levels 2 weeks after injection. For i.v. delivery of DODAC. DOPE-DNA complexes multilamellar vesicles were more effective than large unilamellar vesicles in all organs investigated. Recombinant DNA could be detected in the distal lung region following either route of administration. However, i.t. administration predominantly led to DNA deposition in epithelial cells lining the bronchioles, e.g. in clara cells, whereas i.v. administration resulted in DNA deposition in the alveolar region of the lung including type II alveolar epithelial cells.

Topics: Analysis of Variance; Animals; Autoradiography; Bronchi; Cations; Chloramphenicol O-Acetyltransferase; Cystic Fibrosis; Epithelial Cells; Gene Expression; Gene Transfer Techniques; Genetic Therapy; Immunohistochemistry; Injections, Intravenous; Liposomes; Mice; Mice, Inbred Strains; Phosphatidylethanolamines; Pulmonary Alveoli; Quaternary Ammonium Compounds; Trachea

We compared the transfection efficiency of four types of positively charged liposomes composed of (i) N-(alpha-trimethylammonioacetyl)-didodecyl-D-glutamate chloride (TMAG), dilauroylphosphatidylcholine (DLPC), and dioleoylphosphatidylethanolamine (DOPE) (1:2:2 molar ratio); (ii) 3 beta [N-(N', N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and DOPE (3:2 molar ratio); (iii) dimethyldioctadecylammonium bromide (DDAB) and DOPE (1:2.2 molar ratio); (iv) N-[1-(2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTMA) and DOPE (1:1, w/w; lipofectin). Luciferase gene was used as a reporter gene. Among the cationic lipsomes used, the liposomes composed of TMAG, DOPE and DLPC showed a much higher efficiency of plasmid DNA entrapment than the other cationic liposomes tested. In the absence of serum, the cationic multilamellar vesicles (MLV) and small unilamellar vesicles (SUV) composed of TMAG, DOPE and DLPC gave highly efficient transfection. On the other hand, MLV, dehydration-rehydration vesicles (DRV), and SUV liposomes prepared with the mixtures of DC-Chol and DOPE showed similar levels of transfection efficiency. However, the cationic liposomes composed of DDAB and DOPE showed inferior efficiency, whether in the form of DRV, SUV or MLV. The transfection efficiency of lipofectin was also low. In the presence of serum, on the other hand, a considerable (about 30-50%) amount of transfection activity was still observed at 10% fetal calf serum in the cationic MLV and SUV composed of TMAG, DOPE and DLPC. Cationic MLV, composed of TMAG, DOPE and DLPC, Cationic MLV, composed of TMAG, DOPE and DLPC, can transfect plasmid DNA, not only in the adherent cell lines but also, in the suspension cell lines. These findings indicate that the transfection efficiency of cationic liposomes is affected by the lipid composition, the type of liposome, or the presence or absence of serum. They also indicate that the cationic liposomes containing TMAG, DOPE and DLPC are efficient vectors for gene transfer into cells.

Topics: Animals; Cations; Cats; Cattle; Cell Line; Cholesterol; COS Cells; DNA; Glutamates; Humans; Kidney; Lipids; Liposomes; Mice; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Sheep; Transfection