dioleoyl-phosphatidylethanolamine and didodecyldimethylammonium

We determined the bindings of several lipids such as cholesterol (CHOL), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethyl-ammoniumbromide (DDAB), and dioleoylphosphatidylethanolamine (DOPE) to β-lactoglobulin (β-LG) at physiological conditions. FTIR, CD, and fluorescence spectroscopic methods as well as molecular modeling were used to determine the binding of lipid-protein complexes. Structural analysis showed that lipids bind β-LG via both hydrophilic and hydrophobic interactions with overall binding constants of K(CHOL-β-LG) = 6.0 (±0.6) × 10(3) M(-1), K(DOPE-β-LG) = 6.5 (±0.7) × 10(3) M(-1), K(DDAB-β-LG) = 1.6 (±0.3) × 10(4) M(-1), and K(DOTAP-β-LG) = 2.2 (±0.67) × 10(4) M(-1). The number of lipid bound per protein molecule (n) was 0.8 (CHOL), 0.7 (DOPE), 1.0 (DDAB), and 1.3 (DOTAP). Molecular modeling showed the participation of several amino acid residues in lipid-protein complexation with the order of binding DOTAP > DDAB > DOPE > CHOL. Alterations of the protein conformation were observed in the presence of lipids with a minor decrease in β-sheet and an increase in turn structure.

Topics: Animals; Cations; Cholesterol; Circular Dichroism; Fatty Acids, Monounsaturated; Hydrophobic and Hydrophilic Interactions; Lactoglobulins; Models, Molecular; Phosphatidylethanolamines; Protein Binding; Protein Conformation; Quaternary Ammonium Compounds; Spectrometry, Fluorescence; Spectroscopy, Fourier Transform Infrared

Complexes of cationic liposomes with DNA are promising tools to deliver genetic information into cells for gene therapy and vaccines. Electrostatic interaction is thought to be the major force in lipid-DNA interaction, while lipid-base binding and the stability of cationic lipid-DNA complexes have been the subject of more debate in recent years. The aim of this study was to examine the complexation of calf-thymus DNA with cholesterol (Chol), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethylammoniumbromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE), at physiological condition, using constant DNA concentration and various lipid contents. Fourier transform infrared (FTIR), UV-visible, circular dichroism spectroscopic methods and atomic force microscopy were used to analyse lipid-binding site, the binding constant and the effects of lipid interaction on DNA stability and conformation. Structural analysis showed a strong lipid-DNA interaction via major and minor grooves and the backbone phosphate group with overall binding constants of K(Chol) = 1.4 (+/-0.5) x 10(4) M(-1), K(DDAB) = 2.4 (+/-0.80) x 10(4) M(-1), K(DOTAP) = 3.1 (+/-0.90) x 10(4) M(-1) and K(DOPE) = 1.45 (+/- 0.60) x 10(4) M(-1). The order of stability of lipid-DNA complexation is DOTAP>DDAB>DOPE>Chol. Hydrophobic interactions between lipid aliphatic tails and DNA were observed. Chol and DOPE induced a partial B to A-DNA conformational transition, while a partial B to C-DNA alteration occurred for DDAB and DOTAP at high lipid concentrations. DNA aggregation was observed at high lipid content.

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

We aimed to increase the efficiency of adenoviral vectors by limiting adenoviral spread from the target site and reducing unwanted host immune responses to the vector. We complexed adenoviral vectors with DDAB-DOPE liposomes to form adenovirus-liposomal (AL) complexes. AL complexes were delivered by intratumoral injection in an immunocompetent subcutaneous rat tumor model and the immunogenicity of the AL complexes and the expression efficiency in the tumor and other organs was examined. Animals treated with the AL complexes had significantly lower levels of beta-galactosidase expression in systemic tissues compared to animals treated with the naked adenovirus (NA) (P<0.05). The tumor to non-tumor ratio of beta-galactosidase marker expression was significantly higher for the AL complex treated animals. NA induced significantly higher titers of adenoviral-specific antibodies compared to the AL complexes (P<0.05). The AL complexes provided protection (immunoshielding) to the adenovirus from neutralizing antibody. Forty-seven percent more beta-galactosidase expression was detected following intratumoral injection with AL complexes compared to the NA in animals pre-immunized with adenovirus.. Complexing of adenovirus with liposomes provides a simple method to enhance tumor localization of the vector, decrease the immunogenicity of adenovirus, and provide protection of the virus from pre-existing neutralizing antibodies.

Topics: Adenoviridae; Animals; Antibody Formation; beta-Galactosidase; Cell Line, Tumor; Genes, Reporter; Genetic Vectors; Humans; Liposomes; Molecular Conformation; Particle Size; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Rats; Salivary Gland Neoplasms; Tissue Distribution; Transduction, Genetic

This paper summarizes preliminary results of combining suicide gene strategy (E. coli cytosine deaminase gene--CD) with immunotherapy (murine interleukin-4 gene) for treatment of experimental B16(F10) melanomas implanted into C57Bl/6 mice. The best therapeutic results, inhibition of tumor growth and prolonged survival time of treated vs. control mice, were obtained when plasmid expression vectors containing therapeutic genes were transferred into mice via DDAB/DOPE cationic liposome carrier on the third or fourth day following inoculation of mice with cancer cells. Extension of survival time has been noted in the case of two-gene therapy (as compared with one-gene therapy) of tumors which originated from cells transfected in vitro with CD gene and which were subsequently injected in vivo with IL-4-secreting cells. However, no improvement of therapeutic effect was obtained in case of mice treated with a combination of two genes transferred intratumorally with DDAB/DOPE cationic liposomes as compared to mice treated with a single gene only.

Topics: Animals; Cell Division; Cytosine Deaminase; DNA; Escherichia coli; Genes, Bacterial; Genetic Therapy; Interleukin-4; Liposomes; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Nucleoside Deaminases; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Transfection

1. The effect of liposome phospholipid composition has been assumed to be relatively unimportant because of the presumed inert nature of phospholipids. 2. We have previously shown that cationic liposome formulations used for gene therapy inhibit, through their cationic component, the synthesis by activated macrophages of the pro-inflammatory mediators nitric oxide (NO) and tumour necrosis factor-alpha (TNF-alpha). 3. In this study, we have evaluated the ability of different cationic lipids to reduce footpad inflammation induced by carrageenan and by sheep red blood cell challenge. 4. Parenteral (i.p. or s.c) or local injection of the positively charged lipids dimethyldioctadecylammomium bromide (DDAB), dioleyoltrimethylammonium propane (DOTAP), dimyristoyltrimethylammonium propane (DMTAP) or dimethylaminoethanecarbamoyl cholesterol (DC-Chol) significantly reduced the inflammation observed in both models in a dose-dependent manner (maximum inhibition: 70-95%). 5. Cationic lipids associated with dioleyol- or dipalmitoyl-phosphatidylethanolamine retained their anti-inflammatory activity while cationic lipids associated with dipalmitoylphosphatidylcholine (DPPC) or dimyristoylphosphatidylglycerol (DMPG) showed no anti-inflammatory activity, indicating that the release of cationic lipids into the macrophage cytoplasm is a necessary step for anti-inflammatory activity. The anti-inflammatory activity of cationic lipids was abrogated by the addition of dipalmitoylphosphatidylethanolamine-poly(ethylene)glycol-2000 (DPPE-PEG2000) which blocks the interaction of cationic lipids with macrophages. 6. Because of the significant role of protein kinase C (PKC) in the inflammatory process we have determined whether the cationic lipids used in this study inhibit PKC activity. The cationic lipids significantly inhibited the activity of PKC but not the activity of a non-related protein kinase, PKA. The synthesis of interleukin-6 (IL-6), which is not dependent on PKC activity for its induction in macrophages, was not modified in vitro or in situ by cationic lipids. The synthesis of NO and TNF-alpha in macrophages, both of which are PKC-dependent, was downregulated by cationic lipids. 7. These results demonstrate that cationic lipids can be considered as novel anti-inflammatory agents. The downregulation of pro-inflammatory mediators through interaction of cationic lipids with the PKC pathway may explain this anti-inflammatory activity. Furthermore, since cationic lipids have

Topics: Animals; Anti-Inflammatory Agents; Carrageenan; Cations; Cholesterol; Edema; Erythrocytes; Fatty Acids, Monounsaturated; Female; Liposomes; Mice; Phosphatidylethanolamines; Phospholipids; Protein Kinase C; Quaternary Ammonium Compounds; Sheep

Cationic liposomes improve the delivery of antisense oligonucleotides (ODNs) into cells. However, there is marked variability in the cellular uptake of ODNs into different cell lines. We used liposomes containing dimethyloctadecylammonium bromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE) to increase the delivery of phosphodiester ODNs into four different myeloma cell lines. The delivery by cationic liposomes increased the delivery of bcl-2 antisense ODNs by a factor of 9 to 45 as compared to plain ODNs. The stability of ODNs was increased with liposomes both in the culture medium and within the cells. Intact liposomal ODNs were detected inside the cells up to 24 hours with gel electrophoresis and phosphor imager analysis. Antisense ODNs had no effect on bcl-2 mRNA levels. Also the proliferation of myeloma cells remained unchanged during the 3-day incubation period. Our study shows that liposomal antisense ODNs targeting bcl-2 of human myeloma cells result in increased stability of ODNs with minimal toxicity. However, further modifications are needed to gain biological effects of antisense ODNs on human myeloma cells.

Topics: Cations; Cell Differentiation; Cell Division; Drug Carriers; Drug Stability; Gene Expression Regulation, Neoplastic; Gene Targeting; Genes, bcl-2; Humans; Liposomes; Multiple Myeloma; Oligonucleotides, Antisense; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Tumor Cells, Cultured