Formation Of Non-lamellar Structures Research Articles

Not just a fluidifying effect: omega-3 phospholipids induce formation of non-lamellar structures in biomembranes.

Polyunsaturated omega-3 fatty acid docosahexaenoic acid (DHA) is found in very high concentrations in a few peculiar tissues, suggesting that it must have a specialized role. DHA was proposed to affect the function of the cell membrane and related proteins through an indirect mechanism of action, based on the DHA-phospholipid effects on the lipid bilayer structure. In this respect, most studies have focused on its influence on lipid-rafts, somehow neglecting the analysis of effects on liquid disordered phases that constitute most of the cell membranes, by reporting in these cases only a general fluidifying effect. In this study, by combining neutron reflectivity, cryo-transmission electron microscopy, small angle neutron scattering, dynamic light scattering and electron paramagnetic resonance spectroscopy, we characterize liquid disordered bilayers formed by the naturally abundant 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and different contents of a di-DHA glycero-phosphocholine, 22:6-22:6PC, from both a molecular/microscopic and supramolecular/mesoscopic viewpoint. We show that, below a threshold concentration of about 40% molar percent, incorporation of 22:6-22:6PC in the membrane increases the lipid dynamics slightly but sufficiently to promote the membrane deformation and increase of multilamellarity. Notably, beyond this threshold, 22:6-22:6PC disfavours the formation of lamellar phases, leading to a phase separation consisting mostly of small spherical particles that coexist with a minority portion of a lipid blob with water-filled cavities. Concurrently, from a molecular viewpoint, the polyunsaturated acyl chains tend to fold and expose the termini to the aqueous medium. We propose that this peculiar tendency is a key feature of the DHA-phospholipids making them able to modulate the local morphology of biomembranes.

Cardiolipin plays an essential role in the formation of intracellular membranes in Escherichia coli

Mitochondria, chloroplasts and photosynthetic bacteria are characterized by the presence of complex and intricate membrane systems. In contrast, non-photosynthetic bacteria lack membrane structures within their cytoplasm. However, large scale over-production of some membrane proteins, such as the fumarate reductase, the mannitol permease MtlA, the glycerol acyl transferase PlsB, the chemotaxis receptor Tsr or the ATP synthase subunit b, can induce the proliferation of intra cellular membranes (ICMs) in the cytoplasm of Escherichia coli. These ICMs are particularly rich in cardiolipin (CL). Here, we have studied the effect of CL in the generation of these membranous structures. We have deleted the three genes (clsA, clsB and clsC) responsible of CL biosynthesis in E. coli and analysed the effect of these mutations by fluorescent and electron microscopy and by lipid mass spectrometry. We have found that CL is essential in the formation of non-lamellar structures in the cytoplasm of E. coli cells. These results could help to understand the structuration of membranes in E. coli and other membrane organelles, such as mitochondria and ER.

Biological artificial fluid-induced non-lamellar phases in glyceryl monooleate: the kinetics pathway and its digestive process by bile salts

Background: The cubic (QII) phase is a promising sustained-release system. However, its rigid gel-like propensity is highly viscous, which makes it difficult to handle in pharmaceutical applications. To circumvent this problem, a less viscous lamellar (Lα) phase that could spontaneously transform to QII phase by the introduction of water or biological artificial fluid can be used. However, the kinetics pathway of phase transition, susceptibility to digestive processes and impact of the transition on drug release are not yet well understood.Method: We investigated various biological artificial fluid-induced Lα to inverse QII phase transition over time in glyceryl monooleate (GMO) by water penetration scan and light polarizing microscopy. To reveal the structure stability, fluorescence spectroscopy studies were conducted using pyrene as a probe. Furthermore, the release mechanism of pyrene as a lipophilic drug model in the spontaneously formed QII was investigated.Result: Although hexagonal (HII) mesophases occurred when phosphate buffered saline (PBS) 7.4, 0.1 M HCl or sodium taurocholate (NaTC) solutions were introduced to GMO at room temperature, they disappear with the exception of 0.1 M HCl at 37 °C. Compared with 25 °C, Lα to QII phase transition was in a faster rate as almost completely transforms were observed after 2 h post-immersion. The spontaneously formed mesophases were stable over 24 h immersions in PBS or pancreatic lipase solutions as proven by the extremely low fluorescence signal, however they were digestible by bile salts. This result indicated that digestion by bile salts was the major pathway instead of digestion by lipases. Moreover, pyrene fluorescence spectroscopy confirmed that the digestion by bile salts induced the formation of GMO–bile salt mixed micelles whose performance depended on the bile salt concentrations. This dependence influenced the drug release from the spontaneously formed QII phase.Conclusion: All the results concluded that temperature, pH and ionic strength tendencies for the formation of non-lamellar structures greatly influenced the self-assembly process, thereby affecting the final mesophase structure. The results of this study are important to understand the lamellar to non-lamellar lipid-phase transitions and their possible pharmaceutical applications.

Interactions between pH-sensitive liposomes and model membranes

The structure and dynamics of two different pH-sensitive liposome systems were investigated by means of cryo-transmission electron microscopy and different photophysical techniques. Both systems consisted of dioleoylphosphatidylethanolamine (DOPE) and contained either oleic acid (OA) or a novel acid-labile polyethylene glycol-conjugated lipid (DHCho-MPEG5000) as stabiliser. Proton induced leakage, lipid mixing and structural changes were studied in the absence and presence of EPC liposomes, as well as in the presence of liposomes designed to model the endosome membrane. Neither DHCho-MPEG5000- nor OA-stabilised liposomes showed any tendency for fusion with pure EPC liposomes or endosome-like liposomes composed of EPC/DOPE/SM/Cho (40/20/6/34 mol.%). Our investigations showed, however, that incorporation of lipids from the pH-sensitive liposomes into the endosome membrane may lead to increased permeability and formation of non-lamellar structures. Taken together the results suggest that the observed ability of DOPE-containing liposomes to mediate cytoplasmic delivery of hydrophilic molecules cannot be explained by a mechanism based on a direct, and non-leaky, fusion between the liposome and endosome membranes. A mechanism involving destabilisation of the endosome membrane due to incorporation of DOPE, seems more plausible.

Phospholipase C hydrolysis of phospholipids in bilayers of mixed lipid compositions.

Phosphatidylcholine phospholipase C (EC 3.1.4.3) from Bacillus cereus has been assayed with substrates in the form of large unilamellar vesicles. Phosphatidylcholine, phosphatidylethanolamine (also a substrate for the enzyme), sphingomyelin, and cholesterol have been mixed in various proportions, in binary, ternary, and quaternary mixtures. A lag period, followed by a burst of enzyme activity, has been found in all cases. The activity burst was always accompanied by an increase in turbidity of the vesicle suspension. Varying lipid compositions while keeping constant all the other parameters leads to a range of lag times extending over 2 orders of magnitude (from 0.13 to 38.0 min), and a similar variability is found in maximal enzyme rates (from 0.40 to 55.9 min-1). Meanwhile, the proportion of substrate that is hydrolyzed during the lag period remains relatively constant at 0.10% moles of total lipid, in agreement with the idea that enzyme activation is linked to vesicle aggregation through diacylglycerol-rich patches. Phosphatidylethanolamine and cholesterol enhance the enzyme activity in a dose-dependent way: they reduce the lag times and increase the maximal rates. The opposite is true of sphingomyelin. These lipids exert each its own peculiar effect, positive or negative, either alone or in combination, so that the susceptibility of a given mixture to the enzyme activity can be to some extent predicted from its composition. Phospholipase C activity is not directly influenced by the formation of nonlamellar structures. However, the presence of lipids with a tendency to form nonlamellar phases, such as phosphatidylethanolamine or cholesterol, stimulates the enzyme even under conditions at which purely lamellar phases exist. Conversely sphingomyelin, a well-known stabilizer of the lamellar phase, inhibits the enzyme. Thus phospholipase C appears to be regulated by the overall geometry and composition of the bilayer.