Michel Vandenbranden

The role of endosome destabilizing activity in the gene transfer process mediated by cationic lipids

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′‐tetradeccyl‐propionamidine, 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 dioleylphosphatid‐ylethanolamine (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.

Cationic liposomal lipids: from gene carriers to cell signaling

Cationic lipids are positively charged amphiphilic molecules which, for most of them, form positively charged liposomes, sometimes in combination with a neutral helper lipid. Such liposomes are mainly used as efficient DNA, RNA or protein carriers for gene therapy or immunization trials. Over the past decade, significant progress has been made in the understanding of the cellular pathways and mechanisms involved in lipoplex-mediated gene transfection but the interaction of cationic lipids with cell components and the consequences of such an interaction on cell physiology remains poorly described. The data reported in the present review provide evidence that cationic lipids are not just carriers for molecular delivery into cells but do modify cellular pathways and stimulate immune or anti-inflammatory responses. Considering the wide number of cationic lipids currently available and the variety of cellular components that could be involved, it is likely that only a few cationic lipid-dependent functions have been identified so far.

Cationic lipids activate intracellular signaling pathways

Cationic liposomes are commonly used as a transfection reagent for DNA, RNA or proteins and as a co-adjuvant of antigens for vaccination trials. A high density of positive charges close to cell surface is likely to be recognized as a signal of danger by cells or contribute to trigger cascades that are classically activated by endogenous cationic compounds. The present review provides evidence that cationic liposomes activate several cellular pathways like pro-apoptotic and pro-inflammatory cascades. An improved knowledge of the relationship between the cationic lipid properties (nature of the lipid hydrophilic moieties, hydrocarbon tail, mode of organization) and the activation of these pathways opens the way to the use and design of cationic tailored for a specific application (e.g. for gene transport or as adjuvants).

Orientation into the lipid bilayer of an asymmetric amphipathic helical peptide located at the N-terminus of viral fusion proteins.

The complete amino-acid sequence of viral fusion proteins has been analyzed by the Eisenberg procedure. The region surrounding the cleavage site contains a highly hydrophilic region immediately followed by a membrane-like region. Since the effective cleavage between these two domains seems required to expose the fusogenic domain (located at the N-terminal sequence of the transmembrane like region) which is assumed to interact with the lipid membrane of the host cell, we have focused our analysis on the conformation and mode of insertion of this membrane-like domain in a lipid monolayer. It was inserted as an alpha-helical structure into a dipalmitoylphosphatidylcholine (DPPC) monolayer and its orientation at the lipid/water interface was determined using a theoretical analysis procedure allowing the assembly of membrane components. For each viral protein sequence these N-terminal helical segments oriented obliquely with respect to the lipid/water interface. This rather unusual orientation is envisaged as a prerequisite to membrane destabilization and fusogenic activity.

Orientation and structure of the NH2-terminal HIV-1 gp41 peptide in fused and aggregated liposomes.

For several retroviruses, the N-terminal hydrophobic sequence of the viral envelope glycoprotein has been shown to play a crucial role in the interaction between the virus and the host cell membrane. We report here on the interaction of a synthetic 16 residues peptide corresponding to the gp41 NH2-terminal sequence of Human Immunodeficiency Virus with the phospholipid bilayer. Fluorescence energy transfer measurements show that this peptide can induce lipid mixing of large unilamellar vesicles (LUV) of various compositions at pH 7.4 and 37 degrees C. LUV undergo fusion, provided they contained phosphatidylethanolamine (PE) in their lipid composition. To provide insight into the mechanism of the fusion event, the peptide secondary structure and orientation in the lipid bilayer were determined using Fourier Transform Infrared Spectroscopy (FTIR). The peptide adopts mainly a beta-sheet conformation in the absence of lipids. After interaction with LUV the beta-sheet is partly converted into alpha-helix. The orientation of the peptide with respect to the lipid acyl chains depends on the presence of PE in the lipid bilayer. The peptide is inserted into the lipid bilayer with the helix axis oriented parallel to the lipid acyl chains in the fused vesicles, whereas it is adsorbed parallel to the lipid/water interface in the aggregated vesicles. The role of the two kinds of orientation during the fusion event is discussed.

Fusogenic activity of SIV (Simian Immunodeficiency Virus) peptides located in the GP32 NH2 terminal domain

Peptides of 12, 16 and 24 amino acids length corresponding to the NH2 terminal sequence of SIV gp32 were synthesized. Fluorescence energy transfer studies have shown that those peptides can induce lipid mixing of SUV (Small Unilamellar Vesicles) of various compositions at pH 7.4 and 37 degrees C. LUV (Large Unilamellar Vesicles) were shown to undergo fusion, provided they contained PE in their lipid composition. This work is an attempt to determine how the fusogenic activity depends on the structure of the peptide inserted into a lipidic environment. The peptides secondary structure and orientation in the lipid bilayer were determined using Fourier Transform infrared spectroscopy (FTIR). They adopt mainly a beta-sheet conformation in the absence of lipids. After interaction with DOPC SUV, the beta-sheet is partly converted into alpha-helix oriented obliquely with respect to the membrane interface. We bring here evidence that this oblique orientation is a prerequisite to the fusion process.

Adriamycin inhibits the formation of non-bilayer lipid structures in cardiolipin-containing model membranes

The effect of adriamycin on cardiolipin-containing model membrane systems have been studied by 31P-NMR, freeze-fracture electron microscopy and binding experiments. Adriamycin effectively inhibits the formation of non-bilayer lipid structures induced by Ca2+ and cytochrome c in cardiolipin-containing liposomes. This drug also strongly inhibits the uptake of Ca2+ by cardiolipin into an organic phase. These results are discussed in relation to the cardiotoxic effect of adriamycin and the possible importance of non-bilayer lipid structures for the functioning of the mitochondrion.

DiC14-amidine cationic liposomes stimulate myeloid dendritic cells through Toll-like receptor 4.

DiC14‐amidine cationic liposomes were recently shown to promote Th1 responses when mixed with allergen. To further define the mode of action of diC14‐amidine as potential vaccine adjuvant, we characterized its effects on mouse and human myeloid dendritic cells (DC). First, we observed that, as compared with two other cationic liposomes, only diC14‐amidine liposomes induced the production of IL‐12p40 and TNF‐α by mouse bone marrow‐derived DC. DiC14‐amidine liposomes also activated human DC, as shown by synthesis of IL‐12p40 and TNF‐α, accumulation of IL‐6, IFN‐β and CXCL10 mRNA, and up‐regulation of membrane expression of CD80 and CD86. DC stimulation by diC14‐amidine liposomes was associated with activation of NF‐κB, ERK1/2, JNK and p38 MAP kinases. Finally, we demonstrated in mouse and human cells that diC14‐amidine liposomes use Toll‐like receptor 4 to elicit both MyD88‐dependent and Toll/IL‐1R‐containing adaptor inducing interferon IFN‐β (TRIF)‐dependent responses.

Identification of human plasma proteins that bind to cationic lipid/DNA complex and analysis of their effects on transfection efficiency: implications for intravenous gene transfer.

Interaction of cationic lipid/DNA complex with the plasma is a limiting step for the cationic lipid-mediated intravenous gene transfer and expression process. Most of the plasma components that interact with the complex and inhibit its transfection efficiency are still unknown. In the present work, human plasma proteins and lipoproteins that bind to a cationic lipid/DNA complex were isolated on a sucrose density gradient and identified by 2-D gel electrophoresis. Protein binding did not result in complex dissociation or DNA degradation. The effects of several complex-binding plasma components on the transfection efficiency were studied using lung endothelial cells cultured in vitro. Lipoprotein particles caused a drastic loss of the transfection efficiency of the complex. Surprisingly, fibrinogen was found to activate the transfection process. The roles of these complex-binding plasma components on the complex uptake efficiency were quantitatively assessed using radiolabeled plasmid DNA and qualitatively evaluated using fluorescence microscopy. A good correlation was found between the effects of the complex-binding plasma components on the transfection and on cell uptake efficiencies. In contrast to what was generally believed, our data suggest that disruption of the complex does not occur when it is in contact with the plasma and therefore could not be responsible for the loss of transfection activity. Instead, coating of complexes with plasma components seems to be responsible for reduced uptake by cells, which in turn results in reduced transfection.

INTRACELLULAR VISUALIZATION OF BRDU-LABELED PLASMID DNA/CATIONIC LIPOSOME COMPLEXES

Difficulties in specific detection of transfected DNA in cells represent an important limitation in the study of the gene transfer process. We studied the cellular entry and fate of a plasmid DNA complexed with a cationic lipid, Vectamidine (3-tetradecyl-amino-N-tert-butyl-N-tetradecylpropionamidine) in BHK21 cells. To facilitate its detection inside the cells, bromodeoxyuridine (BrdU) was incorporated into plasmid DNA under conditions that minimize plasmid alteration. BrdU was localized in cells incubated with Vectamidine/BrdU-labeled plasmid DNA complexes by immunogold labeling and electron microscopy (EM). Labeling was predominantly associated with aggregated liposome structures at the surface of and inside the cells. EM observations of cells transfected with Vectamidine/DNA complexes showed that the liposome/DNA aggregates accumulate in large vesicles in the cell cytosol. On the other hand, using rhodamine-labeled Vectamidine and revealing BrdU with FITC-conjugated antibodies permitted simultaneous detection in the cells of both components of the complexes with confocal laser scanning microscopy. The DNA and lipids co-localized at the surface of and inside the cells, indicating that the complex is internalized as a whole. Our results show that the BrdU-labeled plasmid DNA detection system can be a useful tool to visualize exogenous DNA entry into cells by a combination of electron and confocal microscopy.