Ilya Koltover

PHASE DIAGRAM, STABILITY, AND OVERCHARGING OF LAMELLAR CATIONIC LIPID-DNA SELF-ASSEMBLED COMPLEXES

Cationic lipid-DNA (CL-DNA) complexes comprise a promising new class of synthetic nonviral gene delivery systems. When positively charged, they attach to the anionic cell surface and transfer DNA into the cell cytoplasm. We report a comprehensive x-ray diffraction study of the lamellar CL-DNA self-assemblies as a function of lipid composition and lipid/DNA ratio, aimed at elucidating the interactions determining their structure, charge, and thermodynamic stability. The driving force for the formation of charge-neutral complexes is the release of DNA and lipid counterions. Negatively charged complexes have a higher DNA packing density than isoelectric complexes, whereas positively charged ones have a lower packing density. This indicates that the overcharging of the complex away from its isoelectric point is caused by changes of the bulk structure with absorption of excess DNA or cationic lipid. The degree of overcharging is dependent on the membrane charge density, which is controlled by the ratio of neutral to cationic lipid in the bilayers. Importantly, overcharged complexes are observed to move toward their isoelectric charge-neutral point at higher concentration of salt co-ions, with positively overcharged complexes expelling cationic lipid and negatively overcharged complexes expelling DNA. Our observations should apply universally to the formation and structure of self-assemblies between oppositely charged macromolecules.

Structure and Structure—Function Studies of Lipid/Plasmid DNA Complexes

Abstract Recent synchrotron-based X-ray diffraction studies have enabled us to comprehensively solve the self-assembled structures in mixtures of cationic liposomes (CLs) complexed with linear λ-DNA. In one case the CL-DNA complexes were found to consist of a higher ordered multilamellar structure (labeled LCα with DNA sandwiched between cationic bilayer membranes. The membrane charge density is found to control the DNA interaxial spacing with high densities leading to high DNA compaction between lipid bilayers. A second self-assembled structure (labeled HCII) consists of linear DNA strands coated by cationic lipid monolayers and arranged on a 2D hexagonal lattice. In this paper we report on a combined X-ray diffraction and optical microscopy study of CLs complexed with functional supercoiled plasmid DNA. We describe the self-assembled structures in cell culture medium for both a high transfectant complex (DOTAP/DOPE, ΦDOPC = 0.72) and a low transfectant complex (DOTAP/DOPC, ΦDOPC = 0.72). Fluorescence optica microscopy shows two distinct interactions between these two types of complexes and mouse fibroblast L-cells, demonstrating the existence of a correlation between structure and transfection efficiency.

DNA at membrane surfaces : an experimental overview

Atomic force microscopy studies of DNA attached to rigid surfaces were initially motivated by the development-of methods which would stretch DNA for the purposes of rapid sequencing. Currently there is much interest in studies of multilayers of DNA chains self-assembled on membranes which form spontaneously when DNA adsorbs onto oppositely charged cationic liposomes (CLs). A major motivation for elucidating the structures and interactions in these CL-DNA complexes arises for two reasons. The first is that they are known to mimic certain characteristics of viruses by being efficient chemical carriers of genes (DNA sections) for delivery in cells. The second is that they are models of studies of DNA condensation phases in two dimensions. DNA-membrane interactions should also provide clues for the relevant molecular forces in the condensation of DNA in chromosomes and viral capsids. The particular complexes described in this review contain linear DNA forming a new ‘hybrid’ phase of matter; that is, the DNA chains form a finite size two dimensional smectic coupled to the three dimensional smectic phase of membranes.

Structure under confinement in a smectic-A and lyotropic surfactant hexagonal phase

Abstract Simple and complex fluids undergo significant changes in their structural and rheological behaviour as they are progressively confined between narrowing walls. The understanding of these new fluid properties is of fundamental interest in applications ranging from thin film lubrication in micromachines to catalysis. The X-Ray surface forces apparatus (XSFA) is capable of non-destructive imaging of the structure of confined complex fluid systems, on length scales ranging from nanometers to several tens of microns. In this article, recent work is presented on studies of confined complex fluids with the XSFA. Confinement can significantly align the liquid crystalline smectic phase. The degree of orientation depends critically on the compliance of the confining surface: “soft surfaces” exhibit a critical gap for alignment of 3.4 μm, while “hard surfaces” do not exhibit gap dependent alignment. Shear-induced orientation has been shown to dominate over confinement induced alignment with the systems studied. In the final part of the paper, we discuss an important new development where it is demonstrated that the XSFA may be used as an alignment tool for lyotropic liquid crystals.

Study of flow in a smectic liquid crystal in the X-ray Surface Forces Apparatus

AbstractWe describe the newly invented X-Ray Surface Forces Apparatus (X-SFA) which allows the simultaneous measurement of forces and collective structures of confined complex fluids under static and flow conditions. The structure of the smectic liquid crystal 8CB (4-cyano-4′-octylbiphenyl) f confined between two mica surfaces with separation ranging from 4000 to 20,000 A was measured. At small gaps and no shear, the smectic layers take on distinct stable orientations, including the bulk forbidden “h” orientation. which persist under low shear (

Self-Assembled Structures of Lipid/DNA Nonviral Gene Delivery Systems from Synchrotron X-Ray Diffraction

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Structure of DNA-cationic liposome complexes : DNA intercalation in multilamellar membranes in distinct interhelical packing regimes

⩽ 30 s−1). However, at higher shear rates