Biomedical subjects

C R Safinya

Publications and source records attributed to C R Safinya.

17 recordsLinked to original sources

PubMed2005

Microchannel systems in titanium and silicon for structural and mechanical studies of aligned protein self-assemblies.

We report a technique for the alignment of self-assembled protein systems, such as F-actin bundles and microtubules, in a surface-modified titanium or silicon microfluidic device. Assembling filamentous protein systems in a confined geometry produces highly aligned samples for structural and mechanical studies. Biomolecular self-assembly can be investigated in a controlled fashion under different molecular concentration gradients and conditions along a channel length. We have shown that surface-modified devices produced via a high aspect ratio etch process in titanium and silicon can be used to confine and control such macromolecular assemblies and present examples of F-actin bundles and microtubules in this system.

Actins↗

PubMed2004

Skin layer at the actin-gel surface: quenched protein membranes form flat, crumpled, and tubular morphologies.

We report the discovery of a hierarchically structured skin layer formed at the surface of an isotropic gel of filamentous actin bundles at high molar ratios of alpha-actinin, an actin cross-linker, to globular actin. Confocal microscopy has elucidated the full, micron scale 3D structure. The protein skin layer, composed of a directed network of bundles, exhibits flat, crumpled, tubelike and pleated multitubular morphologies, resulting from stresses due to the underlying gel. The skin layer, which readily detaches, constitutes a model anisotropic solid membrane with stress-induced, quenched disorder.

Actin Cytoskeleton↗

PubMed2003

Structure of actin cross-linked with alpha-actinin: a network of bundles.

Three-dimensional laser scanning confocal microscopy has revealed that filamentous actin, when complexed with the cross-linking protein alpha-actinin, will spontaneously assemble on a micron scale into a structure comprised of a relatively rigid, frequently branching, 3D network of bundles with characteristic mesh size of the order of the persistence length of F-actin. In contrast, additional nanoscale ordering is observed, as synchrotron x-ray diffraction has revealed a disordered, distorted square lattice of actin fibers within the individual bundles.

Actin Cytoskeleton↗

PubMed2003

Structural polymorphism of DNA-dendrimer complexes.

DNA condensation in vivo relies on electrostatic complexation with small cations or large histones. We report a synchrotron x-ray study of the phase behavior of DNA complexed with synthetic cationic dendrimers of intermediate size and charge. We encounter unexpected structural transitions between columnar mesophases with in-plane square and hexagonal symmetries, as well as liquidlike disorder. The isoelectric point is a locus of structural instability. A simple model is proposed based on competing long-range electrostatic interactions and short-range entropic adhesion by counterion release.

Cations↗

PubMed2001

Direct observation of shear-induced orientational phase coexistence in a lyotropic system using a modified x-ray surface forces apparatus.

The second generation x-ray surface forces apparatus (XSFA-II) allows for the first time simultaneous in situ small-angle x-ray scattering and surface force measurements. We have used the XSFA-II to monitor shear-induced orientational transitions in a lyotropic model lubricant system. Upon applying small shear amplitudes (approximately 20 micrometer) to a relatively thick (approximately 800 micrometer) film, we observed evidence for the formation of an orientational boundary layer at the shearing surface. Time-resolved x-ray diffraction revealed the gradual transition to shear-favored orientation by growth of the boundary layer.

Journal Article↗

PubMed2001

Structures of lipid-DNA complexes: supramolecular assembly and gene delivery.

Recently, there has been a flurry of experimental work on understanding the supramolecular assemblies that are formed when cationic liposomes (CLs) are mixed with DNA. From a biomedical point of view, CLs (vesicles) are empirically known to be carriers of genes (sections of DNA) in nonviral gene delivery applications. Although viral-based carriers of DNA are presently the most common method of gene delivery, nonviral synthetic methods are rapidly emerging as alternative carriers, because of their ease of production and nonimmunogenicity (viral carriers very often evoke an undesirable and potentially lethal immune response). At the moment, cationic-lipid-based carriers have emerged as the most popular nonviral method to deliver genes in therapeutic applications, for example, CL carriers are used extensively in clinical trials worldwide. However, because the mechanism of transfection (the transfer of DNA into cells by CL carriers, followed by expression) of CL--DNA complexes remains largely unknown, the measured efficiencies are, at present, very low. The low transfection efficiencies of current nonviral gene delivery methods are the result of poorly understood transfection-related mechanisms at the molecular and self-assembled levels. Recently, work has been carried out on determining the supramolecular structures of CL--DNA complexes by the quantitative technique of synchrotron X-ray diffraction. When DNA is mixed with CLs (composed of mixtures of cationic DOTAP and neutral DOPC lipids), the resulting CL--DNA complex consists of a multilamellar structure (L(alpha)(C)) comprising DNA monolayers sandwiched between lipid bilayers. The existence of a different columnar inverted hexagonal (H(II)(C)) phase in CL--DNA complexes was also demonstrated using synchrotron X-ray diffraction. Ongoing functional studies and optical imaging of cells are expected to clarify the relationship between the supramolecular structures of CL--DNA complexes and transfection efficiency.

Animals↗

PubMed2000

DNA condensation in two dimensions.

We have found that divalent electrolyte counterions common in biological cells (Ca(2+), Mg(2+), and Mn(2+) ) can condense anionic DNA molecules confined to two-dimensional cationic surfaces. DNA-condensing agents in vivo include cationic histones and polyamines spermidine and spermine with sufficiently high valence (Z) 3 or larger. In vitro studies show that electrostatic forces between DNA chains in bulk aqueous solution containing divalent counterions remain purely repulsive, and DNA condensation requires counterion valence Z >/= 3. In striking contrast to bulk behavior, synchrotron x-ray diffraction and optical absorption experiments show that above a critical divalent counterion concentration the electrostatic forces between DNA chains adsorbed on surfaces of cationic membranes reverse from repulsive to attractive and lead to a chain collapse transition into a condensed phase of DNA tethered by divalent counterions. This demonstrates the importance of spatial dimensionality to intermolecular interactions where nonspecific counterion-induced electrostatic attractions between the like-charged polyelectrolytes overwhelm the electrostatic repulsions on a surface for Z = 2. This new phase, with a one-dimensional counterion liquid trapped between DNA chains at a density of 0.63 counterions per DNA bp, represents the most compact state of DNA on a surface in vitro and suggests applications in high-density storage of genetic information and organo-metallic materials processing.

Bacteriophage lambda↗

PubMed2000

Hierarchical self-assembly of F-actin and cationic lipid complexes: stacked three-layer tubule networks.

We describe a distinct type of spontaneous hierarchical self-assembly of cytoskeletal filamentous actin (F-actin), a highly charged polyelectrolyte, and cationic lipid membranes. On the mesoscopic length scale, confocal microscopy reveals ribbonlike tubule structures that connect to form a network of tubules on the macroscopic scale (more than 100 micrometers). Within the tubules, on the 0.5- to 50-nanometer length scale, x-ray diffraction reveals an unusual structure consisting of osmotically swollen stacks of composite membranes with no direct analog in simple amphiphilic systems. The composite membrane is composed of three layers, a lipid bilayer sandwiched between two layers of actin, and is reminiscent of multilayered bacterial cell walls that exist far from equilibrium. Electron microscopy reveals that the actin layer consists of laterally locked F-actin filaments forming an anisotropic two-dimensional tethered crystal that appears to be the origin of the tubule formation.

Actins↗

PubMed2000

Structure and structure-function studies of lipid/plasmid DNA complexes.

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 lambda-DNA. In one case the CL-DNA complexes were found to consist of a higher ordered multilamellar structure (labeled L(alpha)C 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 H(II)C) 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, phiDOPE = 0.72) and a low transfectant complex (DOTAP/DOPC, (phiDOPC = 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.

Animals↗

PubMed1999

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.

Biophysical Phenomena↗

PubMed1998

An inverted hexagonal phase of cationic liposome-DNA complexes related to DNA release and delivery.

A two-dimensional columnar phase in mixtures of DNA complexed with cationic liposomes has been found in the lipid composition regime known to be significantly more efficient at transfecting mammalian cells in culture compared to the lamellar (LalphaC) structure of cationic liposome-DNA complexes. The structure, derived from synchrotron x-ray diffraction, consists of DNA coated by cationic lipid monolayers and arranged on a two-dimensional hexagonal lattice (HIIC). Two membrane-altering pathways induce the LalphaC --> HIIC transition: one where the spontaneous curvature of the lipid monolayer is driven negative, and another where the membrane bending rigidity is lowered with a new class of helper-lipids. Optical microscopy revealed that the LalphaC complexes bind stably to anionic vesicles (models of cellular membranes), whereas the more transfectant HIIC complexes are unstable and rapidly fuse and release DNA upon adhering to anionic vesicles.

Birefringence↗

PubMed1998

The influence of polymer molecular weight in lamellar gels based on PEG-lipids.

We report x-ray scattering, rheological, and freeze-fracture and polarizing microscopy studies of a liquid crystalline hydrogel called Lalpha,g. The hydrogel, found in DMPC, pentanol, water, and PEG-DMPE mixtures, differs from traditional hydrogels, which require high MW polymer, are disordered, and gel only at polymer concentrations exceeding an "overlap" concentration. In contrast, the Lalpha,g uses very low-molecular-weight polymer-lipids (1212, 2689, and 5817 g/mole), shows lamellar order, and requires a lower PEG-DMPE concentration to gel as water concentration increases. Significantly, the Lalpha,g contains fluid membranes, unlike Lbeta' gels, which gel via chain ordering. A recent model of gelation in Lalpha phases predicts that polymer-lipids both promote and stabilize defects; these defects, resisting shear in all directions, then produce elasticity. We compare our observations to this model, with particular attention to the dependence of gelation on the PEG MW used. We also use x-ray lineshape analysis of scattering from samples spanning the fluid-gel transition to obtain the elasticity coefficients kappa and B; this analysis demonstrates that although B in particular depends strongly on PEG-DMPE concentration, gelation is uncorrelated to changes in membrane elasticity.

Biophysical Phenomena↗

PubMed1997

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

Cationic liposomes complexed with DNA (CL-DNA) are promising synthetically based nonviral carriers of DNA vectors for gene therapy. The solution structure of CL-DNA complexes was probed on length scales from subnanometer to micrometer by synchrotron x-ray diffraction and optical microscopy. The addition of either linear lambda-phage or plasmid DNA to CLs resulted in an unexpected topological transition from liposomes to optically birefringent liquid-crystalline condensed globules. X-ray diffraction of the globules revealed a novel multilamellar structure with alternating lipid bilayer and DNA monolayers. The lambda-DNA chains form a one-dimensional lattice with distinct interhelical packing regimes. Remarkably, in the isoelectric point regime, the lambda-DNA interaxial spacing expands between 24.5 and 57.1 angstroms upon lipid dilution and is indicative of a long-range electrostatic-induced repulsion that is possibly enhanced by chain undulations.

Bacteriophage lambda↗

PubMed1996

Lamellar biogels: fluid-membrane-based hydrogels containing polymer lipids.

A class of lamellar biological hydrogels comprised of fluid membranes of lipids and surfactants with small amounts of low molecular weight poly(ethylene glycol)-derived polymer lipids (PEG-lipids) were studied by x-ray diffraction, polarized light microscopy, and rheometry. In contrast to isotropic hydrogels of polymer networks, these membrane-based birefringent liquid crystalline biogels, labeled L-alpha,g, form the gel phase when water is added to the liquid-like lamellar L-alpha phase, which reenters a liquid-like mixed phase upon further dilution. Furthermore, gels with larger water content require less PEG-lipid to remain stable. Although concentrated (approximately 50 weight percent) mixtures of free PEG (molecular weight, 5000) and water do not gel, gelation does occur in mixtures containing as little as 0.5 weight percent PEG-lipid. A defining signature of the L-alpha,g regime as it sets in from the fluid lamellar L-alpha phase is the proliferation of layer-dislocation-type defects, which are stabilized by the segregation of PEG-lipids to the defect regions of high membrane curvature that connect the membranes.

Chemical Phenomena↗

PubMed1994

A phase of liposomes with entangled tubular vesicles.

An equilibrium phase belonging to the family of bilayer liposomes in ternary mixtures of dimyristoylphosphatidylcholine (DMPC), water, and geraniol (a biological alcohol derived from oil-soluble vitamins that acts as a cosurfactant) has been identified. Electron and optical microscopy reveal the phase, labeled Ltv, to be composed of highly entangled tubular vesicles. In situ x-ray diffraction confirms that the tubule walls are multilamellar with the lipids in the chain-melted state. Macroscopic observations show that the Ltv phase coexists with the well-known L4 phase of spherical vesicles and a bulk L alpha phase. However, the defining characteristic of the Ltv phase is the Weissenberg rod climbing effect under shear, which results from its polymer-like entangled microstructure.

Acyclic Monoterpenes↗

PubMed1993

Existence of a flat phase in red cell membrane skeletons.

Biomolecular membranes display rich statistical mechanical behavior. They are classified as liquid in the absence of shear elasticity in the plane of the membrane and tethered (solid) when the neighboring molecules or subunits are connected and the membranes exhibit solid-like elastic behavior in the plane of the membrane. The spectrin skeleton of red blood cells was studied as a model tethered membrane. The static structure factor of the skeletons, measured by small-angle x-ray and light scattering, was fitted with a structure factor predicted with a model calculation. The model describes tethered membrane sheets with free edges in a flat phase, which is a locally rough but globally flat membrane configuration. The fit was good for large scattering vectors. The membrane roughness exponent, zeta, defined through h alpha L zeta, where h is the average amplitude of out-of-plane fluctuations and L is the linear membrane dimension, was determined to be 0.65 +/- 0.10. Computer simulations of model red blood cell skeletons also showed this flat phase. The value for the roughness exponent, which was determined from the scaling properties of membranes of different sizes, was consistent with that from the experiments.

Chemical Phenomena↗

PubMedDate not supplied

Synthesis of novel cationic poly(ethylene glycol) containing lipids.

In this paper, the synthesis of novel divalent cationic lipids with poly(ethylene glycol) segments is described. The lipids consist of an unsaturated double-chain hydrophobic moiety based on 3, 4-dihydroxy benzoic acid, attached to a hydrophilic poly(ethylene glycol) spacer which contains a divalent cationic end group. As poly(ethylene glycol) spacers monodisperse triethylene glycol and telechelic poly(ethylene glycol)s with an average degree of polymerization of 9, 23, and 45 were used. The divalent cationic end group was attached by coupling a protected dibasic amino acid to the PEG spacer and following cleavage of the protecting groups. These novel class of cationic lipids is of particular interest for nonviral gene delivery applications.

Hydroxybenzoates↗