Probing the in vitro mechanism of action of cationic lipid/DNA lipoplexes at a nanometric scale.

Cationic lipids are used for delivering nucleic acids (lipoplexes) into cells for both therapeutic and biological applications. A better understanding of the identified key-steps, including endocytosis, endosomal escape and nuclear delivery is required for further developments to improve their efficacy. Here, we developed a labelling protocol using aminated nanoparticles as markers for plasmid DNA to examine the intracellular route of lipoplexes in cell lines using transmission electron microscopy. Morphological changes of lipoplexes, membrane reorganizations and endosomal membrane ruptures were observed allowing the understanding of the lipoplex mechanism until the endosomal escape mediated by cationic lipids. The study carried out on two cationic lipids, bis(guanidinium)-tris(2-aminoethyl)amine-cholesterol (BGTC) and dioleyl succinyl paramomycin (DOSP), showed two pathways of endosomal escape that could explain their different transfection efficiencies. For BGTC, a partial or complete dissociation of DNA from cationic lipids occurred before endosomal escape while for DOSP, lipoplexes remained visible within ruptured vesicles suggesting a more direct pathway for DNA release and endosome escape. In addition, the formation of new multilamellar lipid assemblies was noted, which could result from the interaction between cationic lipids and cellular compounds. These results provide new insights into DNA transfer pathways and possible implications of cationic lipids in lipid metabolism.

Annexin A5-functionalized liposomes for targeting phosphatidylserine-exposing membranes.

Long-circulating liposomes functionalized with cell-targeting elements and loaded with bioactive compounds present high interest as drug delivery nanosystems. We present here the synthesis and physicochemical characterization of liposomes containing PEGylated lipids covalently linked to oriented Annexin-A5 (Anx5) proteins, and we show that Anx5-functionalized liposomes are able to target phosphatidylserine (PS)-exposing membranes. The covalent coupling of Anx5 to liposomes is almost quantitative, which is mainly due to the high accessibility of the reacting groups. The influence of Anx5 functionalization on liposome aggregation was investigated by dynamic light scattering, showing that Anx5-functionalized liposomes are stable below a threshold density of 250 Anx5 molecules per liposome. Anx5-functionalized liposomes bind PS-containing membranes with very high efficacy, which is mainly due to the controlled orientation of the Anx5 at the liposome surface. A striking result, obtained by quartz crystal microbalance with dissipation monitoring, is that one single Anx5 molecule is able to anchor a liposome to a PS-containing supported membrane. Finally, we show by fluorescence microscopy that Anx5-functionalized liposomes bind PS-exposing apoptotic K562 cells with high specificity. This study demonstrates that Anx5-functionalized liposomes bind specifically to PS membranes and are thus potential candidates to deliver drug or imaging agents to sites of apoptosis or thrombosis.

Optimized synthesis of 100 nm diameter magnetoliposomes with high content of maghemite particles and high MRI effect.

Magnetoliposomes are liposomes surrounding an iron oxide core, which are used as contrast enhancing agents in magnetic resonance imaging (MRI). One method for producing magnetoliposomes consists of hydration of a lipid film with citrate-coated iron oxide particles followed by extrusion. Two parameters are of major importance for in vivo applications of magnetoliposomes, namely their size, which must be small, optimally around 100 nm diameter, in order to ensure their prolonged circulation in the bloodstream, and their iron content, which must be maximal for generating high MRI effect. We studied the formation of magnetoliposomes by passive encapsulation of maghemite (γ-Fe(2)O(3)) particle suspensions of varying concentrations, with the objective of producing magnetoliposomes of small size and high iron content. The iron to lipid ratio was used to determine the iron content of the magnetoliposomes after the successive purification steps and cryo-TEM was used to characterize their size, their homogeneity and the efficiency of purification. The size of citrate-coated maghemite clusters was found to be of critical importance for obtaining magnetoliposomes smaller than 200 nm. We were able to reproducibly synthesize magnetoliposomes of 100 nm diameter with high iron content -up to 77 particles per liposome (5.6 moles iron per mole lipid) - and high r(2) MRI relaxivity - up to 320 m m(-1) . s(-1) . The magnetoliposomes present improved characteristics compared with previous reports. Future research will focus on using these magnetoliposomes as drug delivery systems for in vivo diagnostics or therapeutics applications.

Development of a platform of antibody-presenting liposomes.

Antibody-presenting liposomes present high interest as drug delivery systems. The association of antibodies to liposomes is usually realized by covalent coupling of IgGs or their antigen-binding fragments to lipid polar head groups by means of hetero-bifunctional crosslinkers. We present here an original platform of IgG-presenting liposomes which is based on a fusion protein between Annexin-A5 (Anx5) and the IgG-binding ZZ repeat derived from Staphylococcus aureus protein A. The Anx5ZZ fusion protein acts as a bi-functional adaptor that anchors IgGs to liposomes in a non covalent and highly versatile manner. The interactions between IgGs, Anx5ZZ and liposomes were characterized by PAGE, dynamic light scattering and fluorescence quenching assays, establishing that binding of Anx5ZZ to IgGs and of Anx5ZZ-IgG complexes to liposomes is complete with stoichiometric amounts of each species. We found that the sequence of assembly is important and that Anx5ZZ-IgG complexes need to be formed first in solution and then adsorbed to liposomes in order to avoid aggregation. The targeting capacity of Anx5ZZ-IgG-functionalized liposomes was demonstrated by electron microscopy on an ex vivo model system of atherosclerotic plaques. This study shows that the Anx5ZZ adaptor constitutes an efficient platform for functionalizing liposomes with IgGs. This platform may present potential applications in molecular imaging and drug delivery.