Brigitte Sternberg

New structures in complex formation between DNA and cationic liposomes visualized by freeze-fracture electron microscopy

Structures formed during interaction of cationic liposomes and plasmid DNA were studied by freeze—fracture electron microscopy and their morphology was found to be dependent on incubation time and DNA concentration. These structures were formed with liposomes composed of DC‐Chol and DOPE after 30 min incubation at DNA: lipid concentrations encompassing maximal transfection activity. They resembled liposome complexes (meatballs) and additionally bilayer‐covered DNA tubules (spaghetti), whereby the DNA‐tubules were found to be connected to the liposome complexes as well as occurring free in the suspension. At later times and higher DNA‐to‐liposome ratios the complexes grow larger while their membranes become discontinuous, allowing the self‐encapsulation of the DNA. The relative transfection potency of the various morphologically distinct structures is discussed.

Morphology of Cationic Liposome/DNA Complexes in Relation to Their Chemical Composition

AbstractElectron microscopy is used to show the morphology of liposome/DNA complexes as related to their cationic component, the molar ratio of the helper lipid (usually DOPE1), the nature of the DNA-component, as well as the composition of the media. Liposomes made of monovalent cationic amphiphiles adhere and fuse during interaction with negatively charged DNA thereby complexing the DNA. The size of the resulting complexes is depending upon charge neutralization and is smallest at a slightly positive net charge. At molar ratios of DOPE, to the cationic component of ≥ 1.5, hexagonal lipid tubules are formed, especially in media containing high salt concentrations, and even in the control lipid mixture, not interacting with any DNA or oligonucleotide. Complexes, made of plasmid-DNA, monovalent cationic amphiphiles, and DOPE at a lower molar ratio, show additionally to the semifused or fused liposomes a new structure, called spaghetti-like structure, representing a bilayer-coated, supercoiled DNA. Single-s...

The effect of temperature and protein content on the dispersive properties of bacteriorhodopsin from H. halobium in reconstituted DMPC complexes free of endogenous purple membrane lipids: A freeze-fracture electron microscopy study

Abstract Ten reconstituted bilayer complexes of bacteriorhodopsin from Halobacterium halobium and 1,2-dimyristoyl- sn -glycerol-3- phosphocholine with protein/lipid mole ratios of between 1:1440 and 1:67, have been entirely free (less than 0.02%) of endogenous purple membrane phospholipid as judged by 31 P-NMR methods using Triton X-100 and cholate as detergents for solubilization of the protein and reconstitution by detergent dialysis. The nitroxide spin-label Tempo has been employed to determine the bilayer gel to liquid-crystalline phase transition temperature of the reconstituted complexes which was shown to broaden with increasing protein content, but remain centered at 23–24°C. Freeze-fracture electron micrographs of the recombinants showed that the arrangement of the protein particles depended upon the temperature from which the complexes were quenched for study, the protein content of the complexes as well as the rate of freezing. In recombinants quenched from below the bilayer phase transition temperature, protein particles were restricted to areas in the bilayers of low structural order at dilute protein content but randomly dispersed at high protein content, regardless of the rate of sample freezing. When quenched from above the bilayer phase transition temperature, complexes quenched at a faster rate of freezing produced randomly dispersed particles whilst a slower rate of freezing produced areas devoid of protein particles. No indication of the characteristic hexagonal packing of bacteriorhodopsin molecules, as reported for the purple membrane (Blaurock, A.E. and Stoeckenius, W. (1971) Nature, 233, 152–155) or reconstituted complexes still containing endogenous purple membrane phospholipids (Cherry, R.J. et al. (1978) J. Mol. Biol. 121, 282–298), were observed. The results are interpreted in terms of a lipid-mediated promotion for bacteriorhodopsin association into the hexagonal lattice, possibly through association with the negatively charged lipids of the purple membrane at the bilayer surface.

Reversible disc-micellization of dimyristoylphosphatidylcholine bilayers induced by melittin and [ALA-14]melittin

The properties of melittin and a synthetic analogue, [Ala-14]melittin (P14A), in inducing reversible transitions between vesicles and micelles at the liquid-crystalline to gel phase transition temperature (Tm) in complexes with saturated phosphatidylcholines has been studied by deuterium NMR and freeze-fracture electron microscopy (EM). At concentrations between 3 and 5 mol% relative to lipid, each peptide causes reversible micellization of dimyristoylphosphatidylcholine (DMPC) bilayers when the temperature is lowered below Tm. At concentrations of 5 mol% relative to lipid, the peptides induce macroscopic magnetic orientation of DMPC bilayers at temperatures around the centre of the lipid phase transition; at temperatures a few degrees above Tm, magnetic orientation is lost. These effects suggest a progressive phase separation of peptide and lipid on cooling the complexes through the phase transition, resulting in increased vesicle deformability. The rates of gel phase micellization, and of bilayer reformation from micelles at temperatures above Tm, are decreased by 100-fold in P14A:DMPC complexes compared with melittin: DMPC complexes. Freeze-fracture EM indicates that P14A suppresses the formation of the gel phase in DMPC bilayers at temperatures below Tm. EM observations of the time-dependence of the reformation of bilayers from micelles after incubating P14A:DMPC micellar complexes at temperatures above Tm indicate that micelles fuse to form growing bilayer sheets from which multilamellar vesicles eventually form. The presence of intramembranous particles (IP) on the fracture faces of both melittin: DMPC complexes and P14A:DMPC complexes in the fluid phase indicates that under the conditions of the study (50 mM Tris-HCl (pH 7.5), 5 mM EDTA) the peptides are organized as discrete aggregates that penetrate deeply into the bilayer.

Coupling of ligands to liposomes independently of solute entrapment: observations on the formed vesicles

Bovine serum albumin (BSA), employed as a model ligand, was covalently linked (about 16% of the amount used) to small unilamellar vesicles (SUV) composed of phospholipid, cholesterol and N-(p-aminophenyl)stearylamide (APSA) (molar ratios 1:1:0.05). SUV with bound BSA were then used to generate dehydration-rehydration vesicles (DRV) in the presence of tetanus toxoid and/or carboxyfluorescein (CF). Nearly all of the SUV-bound BSA (about 15% of the original amount) was recovered in the multilamellar DRV formed, with a considerable proportion (42-62%) of the ligand becoming available on the outer bilayers. This apparent spatial reorientation of BSA within DRV also caused the entrapped toxoid to shift to some extent to the liposomal surface. There was no significant difference in the z average mean size between DRV with and without coupled BSA (543 and 555 nm diameter, respectively). Percent number diameter distribution data revealed that 71.2 (BSA-free) and 76.4% (BSA-containing DRV) of the vesicles had diameters of about 300-440 and 330-420 nm, respectively. However, in terms of percent mass diameter distribution, 69.5% (BSA-free) and 65.2% (BSA-containing DRV) of the mass was in vesicles with corresponding ranges of diameter of 1381-2975 and 1086-2840 nm. Vesicle size heterogeneity in both preparations was confirmed by freeze-fracture electron microscopy which also indicated that structures with or without bound BSA, were mostly vesicular of the multilamellar type. Judging from CF latency values, ligand-bearing DRV were stable on incubation with blood plasma at 37 degrees C for 24 h. Stability was, however, reduced significantly when the amount of ligand bound was excessive. The present approach allows for the coupling of ligands to and the entrapment of antigens and other labile solutes in liposomes independently, thus avoiding potential damage of such solutes by the coupling reagents.

Physicochemical characterisation of human stratum corneum lipid liposomes.

Liposomes were prepared from an extract of all human stratum corneum lipids (hSCL) and characterised in terms of temperature and the presence of Ca2+ by different physicochemical methods. Vesicle aggregation and lateral phase separation were induced by divalent cations with Ca2+ being more efficient than Mg2+. At 24.1 degrees C, i.e. well below physiological temperatures the suspensions consisted of a lamellar phase and crystalline cholesterol. At and above 37 degrees C, this cholesterol surplus was dissolved in the hSCL membranes. However, melting of the hSCL was not completed up to 60 degrees C. The presence of Ca2+ (> or = 9 mM) induced lateral phase separation and fusion of vesicles into extended multilamellar lipid sheets (MLLS) at and above 32.5 degrees C. Upon a subsequent cooling cycle recrystallisation of cholesterol occurred within the MLLS. Finally, membrane mixing of hSCL liposomes with vesicles made of synthetic lipids was investigated. No mixing was observed between either of DPPE/oleic acid, DPPC/DPPE, DPPC/lyso-PC and hSCL liposomes. Mixtures of DPPC/cholesterol hemisuccinate showed a temperature-dependent membrane mixing behaviour, whilst hSCL liposomes and phosphatidylserine liposomes fused temperature-independently with hSCL liposomes.

Incorporation of vitamin D3-derivatives in liposomes of different lipid types.

Vitamin D3-derivatives are known to be effective in differentiation and proliferation of epidermal cells. However, under certain circumstances, they also may show a hypercalcamic activity which can be a serious limitation in their use for dermatological application. For keeping small the negative side effects of vitamin D3-derivatives and for increasing the drug concentration in the skin we investigated their incorporation in liposomes to optimize their use for psoriasis treatment. The incorporation of vitamin D3-derivatives in liposomes of different lipid composition was studied by HPLC, DSC and freeze-fracture electron microscopy. Incorporation rates of more than 80% of the offered drug were found with significant variations related to the number of hydroxyl-groups in the A-ring, opened or closed B-ring, and some modifications in the side chain of the steroid-molecules. In general, the incorporation rates in egg-PC liposomes have been of about 3% up to 10% higher than in DMPC liposomes. Results, obtained by DSC and freeze-fracture electron microscopy, show a depression of the phase transition of DMPC bilayers by incorporation of vitamin D3 already at a concentration of 10 mol%. These results support the idea that vitamin D3 and its analoga investigated are incorporated into the lipid bilayer modifying there the lipid-lipid interactions.

Car☐yacyl derivatives of cardiolipin as four-tailed hydrophobic anchors for the covalent coupling of hydrophilic proteins to liposomes

Two carboxyacyl derivatives of cardiolipin, O-succinyl- and O-glutarylcardiolipin, were synthesized with the aim of using them as artificial membrane anchors for the immobilization of hydrophilic proteins to liposomes. Four adjacent fatty acid residues can be introduced into a protein with only one single amino group being blocked, by reacting the cardiolipin derivatives with the protein amino groups after carbodiimide activation. alpha-Chymotrypsin, used as a model protein, and modified with on average two molecules of O-succinylcardiolipin was incorporated into liposomes, which had been prepared by different methods, with very high yield. If incorporated in preformed liposomes, the carboxyacyl cardiolipin anchors were also efficient in binding proteins to liposomal surfaces. Up to 350 micrograms chymotrypsin/mumol lipid were coupled to small unilamellar vesicles, preserving reactivity of the enzyme towards specific macromolecular inhibitors. Human IgG could also be bound to anchor-containing liposomes with high protein to lipid coupling ratio as well as high coupling yield.

HUMAN STRATUM CORNEUM LIPID-BASED LIPOSOMES (hSCLLs)

AbstractLiposomes were prepared from total lipids extracted from the stratum corneum of human skin. They were studied with respect to size distribution, morphology, phase transitions and marker efflux. Peculiarities of phase transition and morphology are in keeping with the special lipid composition of the horny layer.

Chapter 14 Lipid-protein interactions in controlled membrane protein array and crystal formation

Publisher Summary Integral membrane protein integrity, both at the functional and at the structural level, is controlled and maintained by the presence of either lipids or detergents. This chapter discusses the importance of various lipids in the formation of higher order structures for diffraction studies of a protein. The tendency of any system is to acquire an equilibrium through a process, or series of processes, such that the disorder of that system is maximized. The entropic disordering is, thus, maximized, whereas to form an ordered system, such as a crystal, the total potential energy of the system needs to be minimized, either through bond formation or interactions that produce a negative free energy. It is in bond formation or other interactions where lipids may play a role in the nucleation of a membrane protein. The chapter describes the effects that lipids have been shown to exert on 2D array and 3D crystal formation of integral membrane proteins.