The formation of non-bilayer structures in total polar lipid extracts of chloroplast membranes

The structural organisation of aqueous dispersions of total membrane lipid extracts of broad bean (Vicia faba) chloroplasts is dependent on pH and the presence of cations. In the absence of inorganic salts, sonicated dispersions of lipid extract in distilled water form smooth, single-shell vesicles approximately 30–50 nm in diameter. Reducing the pH of the dispersions, to neutralise the acidic lipids present in the extract, or the addition of low concentrations of metal cations, leads to the fusion of the vesicles and a partial phase-separation of the non-bilayer forming lipid monogalactosyldiacylglycerol to form spherical inverted micelles similar to those previously reported for binary mixtures of monogalactosyl and digalactosyldiacylglycerol (Biochim. Biophys. Acta 685, 297–306). Increasing concentrations of polyvalent, but not monovalent, cations lead to further structural rearrangements involving the formation of para-crystalline arrays of tubular and spherical inverted micelles. The factors determining the formation of these different structures, and their possible relevance to the structural organisation of the native chloroplast membrane, are discussed.     

Microstructural polymorphism in bovine brain galactocerebroside and its two major subfractions.

Aqueous suspensions of either brain galactocerebrosides or its subfraction consisting of alpha-hydroxyacyl galactocerebrosides are mainly composed of vesicles or granular lipid with occasional multilamellar sheets. In aqueous media the other subfraction consisting of non-hydroxyacyl galactocerebrosides forms some helical structures, but most of the lipid remains as granules or vesicles. It is demonstrated that thermal cycling of non-hydroxyacyl galactocerebrosides in polar nonaqueous solvents can greatly enhance the degree of conversion to helical ribbons about 100 nm in diameter. These structures appear to be a stable dehydrated crystalline form of this lipid and are morphologically similar to helical microstructures produced by a few synthetic lipids. On the other hand, similar treatment of unfractionated bovine brain cerebroside and its alpha-hydroxy fatty acyl subfraction quantitatively produces straight needles that appear to be cochleate cylinders. While their dimensions depend on formation conditions, a typical suspension has uniform particles with diameters close to 100 nm and lengths variable from one to a few hundred micrometers. This is the first report demonstrating the quantitative formation of crystalline high axial ratio microstructures from complex mixtures of natural lipids. The different microstructures formed by the two components appear related to the various forms of lipid deposits occurring in lipid storage diseases. The similarity of these "synthetic" microstructures to biological structures in which they are found (such as myelin and intestinal brush border microvilli) strengthens the possibility that galactocerebrosides have a role in stabilizing cylindrical biological structures.     

Bilayer and non-bilayer transformations in aqueous dispersions of mixed sn-3-galactosyldiacylglycerols isolated from chloroplasts A freeze-fracture study

A number of different particle and ‘particle-like’ structures are observed in freeze-fracture replicas prepared from aqueous dispersions of mixtures of mono- and digalactosyldiacylglycerol. The smallest of these structures (10–12 nm in diameter) corresponding to inverted lipid micelles sandwiched within lipid bilayers are often organised into extensive planar arrays. A number of larger ‘particle-like’ features are also observed in replicas of this type. An analysis of the relationship between these structures suggests that they reflect responses to stresses associated with a temperature-dependent incorporation of the lipids of the inverted micelles into the lamellar structure.     

Effects of reconstitution method on the structural organization of isolated chloroplast membrane lipids

Plant galactolipids were isolated from spinach thylakoids and reconstituted by (1) hydration in water or buffer, (2) solubilization in Triton X-100 and subsequent slow detergent removal, and (3) reverse phase evaporation using Freon 11 (trichlorofluoromethane, b.p. 23°C). Digalactosyldiacylglycerol (DG) formed bilayer liposomes when reconstituted by any of these methods. Monogalactosyldiacylglycerol (MG) was very difficult to transfer quantitatively to the aqueous phase. Reverse phase evaporation was the most successful method, and conventional hydration in water or buffer the least efficient, for reconstituting MG quantitatively. Freeze-fracture electron microscopy of pure MG showed arrays of hexagonal II tubes as well as packed inverted micelles (7–9 nm diameter in both cases) covered by a monolayer of lipid. Reconstitution of binary mixtures of MG and DG using the various methods produced the same structures. However, the concentration of MG at which various structural changes occurred depended on the method used for reconstitution. Some of the differences between buffer-hydrated and reverse phase evaporated-reconstituted DG/MG mixtures were traced to the conventional hydration method leaving MG selectively behind on the glass. Reverse phase evaporation allowed the most MG to be incorporated into vesicular structures (about 50% vs. about 30–40% by the detergent method). Irregularities in the bilayer vesicles, ‘lipidic particles’ and ‘fusion pores’, varied proportionally with the amount of MG in the mixture. The transition from vesicular structures to packed tubes and particles had occured by approx. 66% MG using reverse phase evaporation and by approx. 50% MG using detergent solubilization. Aggregates of packed inverted micelles were present in several DG/MG mixtures. The diameters of the inverted micelles varied from 7–9 nm (pure MG) to 20–21 nm (60:40, DG/MG). A model is presented that relates this variation in diameter geometrically to the overall ‘cone’ shape of an MG molecule and the cylindrical shape of DG. In contrast to a previous report, glycerol had no effect on the type of structures observed in replicas of mixed DG/MG samples. However, all structures were more clearly defined in freeze-fracture replicate from glycerinated samples.     

Liposomes, disks, and spherical micelles: aggregate structure in mixtures of gel phase phosphatidylcholines and poly(ethylene glycol)-phospholipids

Poly(ethylene glycol) (PEG) decorated lipid bilayers are widely used in biomembrane and pharmaceutical research. The success of PEG-lipid stabilized liposomes in drug delivery is one of the key factors for the interest in these polymer/lipid systems. From a more fundamental point of view, it is essential to understand the effect of the surface grafted polymers on the physical-chemical properties of the lipid bilayer. Herein we have used cryo-transmission electron microscopy and dynamic light scattering to characterize the aggregate structure and phase behavior of mixtures of PEG-lipids and distearoylphosphatidylcholine or dipalmitoylphosphatidylcholine. The PEG-lipids contain PEG of molecular weight 2000 or 5000. We show that the transition from a dispersed lamellar phase (liposomes) to a micellar phase consisting of small spherical micelles occurs via the formation of small discoidal micelles. The onset of disk formation already takes place at low PEG-lipid concentrations (<5 mol %) and the size of the disks decreases as more PEG-lipid is added to the lipid mixture. We show that the results from cryo-transmission electron microscopy correlate well with those obtained from dynamic light scattering and that the disks are well described by an ideal disk model. Increasing the temperature, from 25 degrees C to above the gel-to-liquid crystalline phase transition temperature for the respective lipid mixtures, has a relatively small effect on the aggregate structure.     

Ultrastructural Organization of the Cytoplasmic Membrane of Anaerobacter polyendosporusas Evidenced by Electron Microscopic Cryofractography

Anaerobacter polyendosporuscells do not have typical mesosomes. However, the analysis of this anaerobic multispore bacterium by electron microscopic cryofractography showed that its cytoplasmic membrane contains specific intramembrane structures in the form of flat lamellar inverted lipid membranes tenths of nanometers to several microns in size. It was found that these structures are located in the hydrophobic interior between the outer and inner leaflets of the cytoplasmic membrane and do not contain intramembrane particles that are commonly present on freeze-fracture replicas. The flat inverted lipid membranes were revealed in bacterial cells cultivated under normal growth conditions, indicating the existence of a complex-type compartmentalization in biological membranes, which manifests itself in the formation of intramembrane compartments having the appearance of vesicles and inverted lipid membranes.     

Effect of polyethyleneglycol-phospholipids on aggregate structure in preparations of small unilamellar liposomes.

Phospholipids with covalently attached poly(ethylene glycol) (PEG lipids) are commonly used for the preparation of long circulating liposomes. Although it is well known that lipid/PEG-lipid mixed micelles may form above a certain critical concentration of PEG-lipid, little is known about the effects of PEG-lipids on liposome structure and leakage at submicellar concentrations. In this study we have used cryogenic transmission electron microscopy to investigate the effect of PEG(2000)-PE on aggregate structure in preparations of liposomes with different membrane compositions. The results reveal a number of important aggregate structures not documented before. The micrographs show that enclosure of PEG-PE induces the formation of open bilayer discs at concentrations well below those where mixed micelles begin to form. The maximum concentration of PEG-lipid that may be incorporated without alteration of the liposome structure depends on the phospholipid chain length, whereas phospholipid saturation or the presence of cholesterol has little or no effect. The presence of cholesterol does, however, affect the shape of the mixed micelles formed at high concentrations of PEG-lipid. Threadlike micelles form in the absence of cholesterol but adapt a globular shape when cholesterol is present.     

Polyunsaturated fatty acyl residues of galactolipids are involved in the control of bilayer/non-bilayer lipid transitions in higher plant chloroplasts

Total polar lipid extracts of chloroplasts isolated from broad beans (Vicia faba) tend to form non-bilayer structures when dispersed in dilute salt solutions. Monoglactosyldiacylglycerol is shown to play a dominant role in this process. The tendency of this lipid to form non-bilayer structures when dispersed alone in water was found to depend upon the degree of unsaturation of its associated fatty acyl chains. Highly unsaturated lipids (average number of double bonds per lipid molecule greater than about 5.0) form inverted hexagonal (Hex_II) structures in water at 20°C, whilst more saturated lipids (average number of double bonds per lipid molecule less than about 4.5) form lamellar sheets. Wide-angle X-ray diffraction and differential scanning calorimetry measurements indicate that these lamellae consist of gel-phase lipid that can adopt either of two structures depending on the thermal history of the sample. Freeze-fracture studies performed on total polar lipid extracts that have been hydrogenated using Adams' catalyst, and reconstituted extracts in which monogalactosyldiacylglycerol has been selectively hydrogenated, show that the degree of unsaturation of this lipid is a key factor in determining whether or not non-bilayer structures are formed in such extracts. Increasing the extent of saturation of the acyl residues of monogalactosyldiacylglycerol reduces the tendency to form non-bilayer structures. Similar effects are observed on lowering the temperature of the dispersions. Fluorescence polarisation measurements using 1,6-diphenyl-1,3,5-hexatriene indicate that the disappearance of non-bilayer structures is accompanied by a marked decrease in the fluidity of the lipid matrix. The possible significance of these observations is discussed in terms of the thermal adaptation and chilling sensitivity of plant membranes.     

Metallosomes.

Structures and ordered arrays containing organometallic particles have potential application in nanofabrication, smaller computer components, optical devices, sensors, and membrane probes and as detection agents. Here, we describe construction of gold clusters covalently attached to lipids and their use in forming typical lipid structures: micelles, liposomes ("metallosomes"), and sheets on an air-water interface. Two sizes of gold clusters were used, undecagold, with an 11-gold atom core 0.8 nm in diameter, and the larger Nanogold, with a 1.4-nm gold core. The morphology of the structures formed was determined by electron microscopy at a resolution at which single gold-lipid molecules were visualized. Further modification by additional catalytic metal deposition enhanced detectability. The approach is flexible and permits a wide variety of metal particle structures to be created using known lipid structures as templates. Additionally, these gold-lipids may serve as useful membrane labels.     

Phase behaviour of mixtures of lipid X with phosphatidylcholine and phosphatidylethanolamine

The effect of increasing concentrations of lipid X (2,3-bis(3-hydroxymyristoyl)-alpha-D-glucosamine 1-phosphate) on the phase behaviour of EPC (egg phosphatidylcholine) and EPE (egg phosphatidylethanolamine) is studied at a pH greater than or equal to 7 where lipid X carries one to two negative charges. Small amounts of lipid X (molar ratio approximately 0.01) induce continuous swelling of EPC and EPE bilayers and consequently the formation of large unilamellar vesicles in excess water. In many respects, the effect of lipid X on EPC and EPE bilayers is similar to that of phosphatidic acid. However, lipid X/EPC mixtures form micelles in excess lipid X whereas mixtures of phosphatidic acid/EPC vesiculate at all ratios. The same is true for lipid X/EPE mixtures. Small unilamellar vesicles of an average diameter of 40 nm form spontaneously upon dispersion of a dry lipid X/EPE film (molar ratio = 10). Unsonicated dispersions of lipid X/EPC (molar ratio = 1) are subjected to pH-jump treatment which involves raising of the pH to 11-12 and subsequent lowering of the pH to between 7.5 and 8.5. Such a treatment has little effect on the vesicle size and size distribution as compared to a control dispersion at pH 8.2. The mean size is determined to be 92 +/- 60 nm. Electron micrographs of freeze-fractured samples of lipid X/EPC (molar ratio = 1) reveal the presence of mainly micelles at pH 12. Upon lowering the pH to neutrality these micelles become unstable and aggregate/fuse rapidly to unilamellar vesicles (average diameter 95 +/- 40 nm). Sonication of equimolar mixtures of lipid X and EPC at pH 7 yields small unilamellar vesicles of a diameter of 20-25 nm as well as mixed micelles of a size between 15 and 17 nm. This behaviour is again different from that of mixed EPC/phosphatidic acid dispersions which form small unilamellar vesicles. The presence of lipid X in such mixtures does not prevent the aggregation/fusion to larger vesicles during freezing of the dispersion. As with pure EPC bilayers, stabilization is, however, achieved in the presence of 10% sucrose. This indicates that the covalently bonded glucosamine group of lipid X cannot substitute water of hydration in neighbouring EPC molecules.     

Self-assembly of DNA-polymer complexes using template polymerization.

The self-assembly of supramolecular complexes of nucleic acids and polymers is of relevance to several biological processes including viral and chromatin formation as well as gene therapy vector design. We now show that template polymerization facilitates condensation of DNA into particles that are <150 nm in diameter. Inclusion of a poly(ethylene glycol)-containing monomer prevents aggregation of these particles. The DNA within the particles remains biologically active and can express foreign genes in cells. The formation or breakage of covalent bonds has until now not been employed to compact DNA into artificial particles.     

The structure of divalent cation-induced aggregates of PIP2 and their alteration by gelsolin and tau.

Phosphatidylinositol bisphosphate (PIP2) serves as a precursor for diacylglycerol and inositol trisphosphate in signal transduction cascades and regulates the activities of several actin binding proteins that influence the organization of the actin cytoskeleton. Molecules of PIP2 form 6-nm diameter micelles in water, but aggregate into larger, multilamellar structures in physiological concentrations of divalent cations. Electron microscopic analysis of these aggregates reveals that they are clusters of striated filaments, suggesting that PIP2 aggregates form stacks of discoid micelles rather than multilamellar vesicles or inverted hexagonal arrays as previously inferred from indirect observations. The distance between striations within the filaments varies from 4.2 to 5.4 nm and the diameter of the filaments depends on the dehydrated ionic radius of the divalent cation, with average diameters of 19, 12, and 10 nm for filaments formed by Mg2+, Ca2+, and Ba2+, respectively. The structure of the divalent cation-induced aggregates can be altered by PIP2 binding proteins. Gelsolin and the microtubule associated protein tau both affect the formation of aggregates, indicating that tau acts as a PIP2 binding protein in a manner similar to gelsolin. In contrast, another PIP2 binding protein, profilin, does not modify the aggregates.     

Thiol-functionalized polymeric micelles: from molecular recognition to improved mucoadhesion

Surface-modified colloids which can selectively interact with biological species or surfaces show promise as drug delivery systems. However, the preparation of such targeted devices remains challenging, especially when considering polyion complex micelles for which side reactions with the ionic core components (typically carboxylic acid or amino groups) can occur. To solve this issue, an innovative synthetic strategy is proposed and used to prepare an asymmetric poly(ethylene glycol)-block-poly(2-(N,N-dimethylamino)ethyl methacrylate) copolymer presenting a thiol group at the end of the poly(ethylene glycol) chain. Thiol groups are highly appealing given that they react almost exclusively and quantitatively with maleimides under physiological conditions, thereby facilitating the chemical functionalization of the copolymer. The simplicity of the derivatization procedure is illustrated by preparing model biotin-capped copolymers. The biotinylated copolymers are shown to self-assemble with an oligonucleotide in aqueous media to form polyion complex micelles with biotin groups at their outer surface. These micelles are capable of molecular recognition toward streptavidin. Alternatively, thiol-decorated (nonderivatized) micelles are prepared and show improved mucoadhesion through the formation of disulfide bonds with mucin. Finally, intermicellar disulfide bonds are generated under oxidative conditions to promote the formation of stimuli-responsive micellar networks.     

A micellar prodrug of paclitaxel conjugated to cyclotriphosphazene

A novel water soluble and biodegradable cyclotriphosphazene-paclitaxel conjugate was prepared by reacting 2'-succinyl paclitaxel with cyclotriphosphazenes bearing equimolar glycyl-L-lysine and methoxy poly(ethylene glycol) as side groups. The macromolecular conjugate was found to self-assemble in aqueous solution to form stable micelles with a mean hydrodynamic diameter of 24.7 nm and a low critical micelle concentration of 10 mg/L. The present conjugate exhibited lower than free paclitaxel but reasonably high in vitro cytotoxicity against selected human tumor cells due to their hydrolytic degradation in PBS solution.     

Morphology of lipid micelles containing lysolecithin.

Mixtures of lysolecithin with various phospholipids were studied by electron microscopy using negative staining. Mixtures of dipalmitoyllecithin and lysolecithin produced disc-shaped structures which were stacked in aggregates with a 6.0--6.4 nm repeat. The disc were 10--50 nm in diameter. The disc-shaped structures were best observed in equimolar mixtures of dipalmitoyllecithin and lysolecithin. When dipalmitoyllecithin was replaced by dimyristoyllecithin, the structures were rather different from those observed in the system containing dipalmitoyllecithin; a cylindrical micellar phase was predominant. Equimolar mixtures of egg lecithin and lysolecithin formed the more usual smectic, concentric lamellae (liposomes) and elongated rod-like micelles which might be bimolecular fragments of spherules. The radius of the rod-like micelles was about 6 nm. Structures of rod-like micelles were observed more frequently in the samples after incubation at room temperature and then further incubation at 0 degrees C. Equimolar mixtures of didecanoyllecithin and lysolecithin produced large amounts of elongated rod-like micelles. Beef brain sphingoymyelin showed disc-shaped structures when mixed with lysolecithin. Incorporation of cholesterol into the mixtures of dipalmitoyllecithin and lysolecithin changed the morphological structure; the size of the disc became larger and eventually liposomes were formed with an increase of cholesterol content. The structures observed in mixtures of dipalmitoyllecithin or sphingomyelin and lysolecithin closely resembled those observed in complexes of apolipoprotein and lipid.     

LHRH receptor-mediated delivery of siRNA using polyelectrolyte complex micelles self-assembled from siRNA-PEG-LHRH conjugate and PEI

Polyelectrolyte complex (PEC) micelles modified with cancer cell targeting moieties were prepared for intracellular delivery of vascular endothelial growth factor (VEGF) small interfering RNA (siRNA). A luteinizing hormone-releasing hormone (LHRH) peptide analogue was coupled as a cancer targeting ligand to the distal end of the poly(ethylene glycol) (PEG)-siRNA conjugate. The siRNA-PEG-LHRH conjugate self-assembled to form nanosized PEC micelles upon mixing with poly(ethylenimine) (PEI) via ionic interactions. The PEC micelles showed spherical morphology with a hydrodynamic diameter of ca. 150 nm. For LHRH receptor overexpressing ovarian cancer cells (A2780), the PEC micelles with LHRH exhibited enhanced cellular uptake compared to those without LHRH, resulting in increased VEGF gene silencing efficiency via receptor-mediated endocytosis. This study showed that PEC micelles decorated with specific cell-recognizable targeting ligands could be used for targeted delivery of siRNA.     

Targeted albumin-based nanoparticles for delivery of amphipathic drugs

We report the preparation and physical and biological characterization of human serum albumin-based micelles of approximately 30 nm diameter for the delivery of amphipathic drugs, represented by doxorubicin. The micelles were surface conjugated with cyclic RGD peptides to guide selective delivery to cells expressing the α(v)β(3) integrin. Multiple poly(ethylene glycol)s (PEGs) with molecular weight of 3400 Da were used to form a hydrophilic outer layer, with the inner core formed by albumin conjugated with doxorubicin via disulfide bonds. Additional doxorubicin was physically adsorbed into this core to attain a high drug loading capacity, where each albumin was associated with about 50 doxorubicin molecules. The formed micelles were stable in serum but continuously released doxorubicin when incubated with free thiols at concentrations mimicking the intracellular environment. When incubated with human melanoma cells (M21+) that express the α(v)β(3) integrin, higher uptake and longer retention of doxorubicin was observed with the RGD-targeted micelles than in the case of untargeted control micelles or free doxorubicin. Consequently, the RGD-targeted micelles manifested cytotoxicity at lower doses of drug than control micelles or free drug.     

Phase behavior and aggregate structure in mixtures of dioleoylphosphatidylethanolamine and poly(ethylene glycol)-lipids

Cryo-transmission electron microscopy has been used to investigate the phase behavior and aggregate structure in dilute aqueous mixtures of dioleoylphosphatidylethanolamine (DOPE) and poly(ethylene glycol)-phospholipids (PEG-lipids). It is shown that PEG-lipids (micelle-forming lipids) induce a lamellar phase in mixtures with DOPE (inverted hexagonal forming lipid). The amount of PEG-lipid that is needed to induce a pure dispersed lamellar phase, at physiological conditions, depends on the size of the PEG headgroup. In the transition region between the inverted hexagonal phase and the lamellar phase, particles with dense inner textures are formed. It is proposed that these aggregates constitute dispersed cubic phase particles. Above bilayer saturating concentration of PEG-lipid, small disks and spherical micelles are formed. The stability of DOPE/PEG-lipid liposomes, prepared at high pH, against a rapid drop of the pH was also investigated. It is shown that the density of PEG-lipid in the membrane, sufficient to prevent liposome aggregation and subsequent phase transition, depends on the size of the PEG headgroup. Below a certain density of PEG-lipid, aggregation and phase transition occurs, but the processes involved proceed relatively slow, over the time scale of weeks. This allows detailed studies of the aggregate structure during membrane fusion.     

Ultrastructural organization of cytoplasmatic membrane of Anaerobacter polyendosporus studied by electron microscopic cryofractography]

Anaerobacter polyendosporus cells do not have typical mesosomes. However, the analysis of this anaerobic multispore bacterium by electron microscopic cryofractography showed that its cytoplasmic membrane contains specific intramembrane structures in the form of flat lamellar inverted lipid membranes tenths of nanometers to several microns in size. It was found that these structures are located in the hydrophobic interior between the outer and inner leaflets of the cytoplasmic membrane and do not contain intramembrane particles that are commonly present on freeze-fracture replicas. The flat inverted lipid membranes were revealed in bacterial cells cultivated under normal growth conditions, indicating the existence of a complex-type compartmentalization in biological membranes, which manifests itself in the formation of intramembrane compartments having the appearance of vesicles and inverted lipid membranes.     

A new method of vesicle formation by salting-out and its application to the reconstitution of sarcoplasmic reticulum

This paper describes a new method of forming membrane vesicles. It was found that the addition of salt such as KCl into a solution containing lipid (asolectin) and a non-ionic surfactant, Triton X-114, led to the formation of closed membrane vesicles. The vesicles were separated from Triton X-114 by hydrophobic interaction chromatography. Electron microscopy revealed that the mean diameter of the vesicles was 110 nm +/- 69 nm (S.D.). Measurement of osmotic volume change showed that the permeability of the vesicle was very low to salts, sugar (glucose) and amphoteric ion (glycine), but very high to glycerol, ethylene glycol and water. Vesicle formation by this 'salting-out' method is very useful for reconstitution of transport systems in biomembranes because of its advantages: completion within a short time; high yield; and the possibility of utilizing samples in non-ionic surfactant solution. When we applied the method to the reconstitution of sarcoplasmic reticulum, Ca2+-ATPase was incorporated into the reconstituted vesicles and was enzymatically active in the membrane.