Aqueous Phospholipid Dispersions Research Articles

The Chemical Reactivity of Membrane Lipids.

It is well-known that aqueous dispersions of phospholipids spontaneously assemble into bilayer structures. These structures have numerous applications across chemistry and materials science and form the fundamental structural unit of the biological membrane. The particular environment of the lipid bilayer, with a water-poor low dielectric core surrounded by a more polar and better hydrated interfacial region, gives the membrane particular biophysical and physicochemical properties and presents a unique environment for chemical reactions to occur. Many different types of molecule spanning a range of sizes, from dissolved gases through small organics to proteins, are able to interact with membranes and promote chemical changes to lipids that subsequently affect the physicochemical properties of the bilayer. This Review describes the chemical reactivity exhibited by lipids in their membrane form, with an emphasis on conditions where the lipids are well hydrated in the form of bilayers. Key topics include the following: lytic reactions of glyceryl esters, including hydrolysis, aminolysis, and transesterification; oxidation reactions of alkenes in unsaturated fatty acids and sterols, including autoxidation and oxidation by singlet oxygen; reactivity of headgroups, particularly with reactive carbonyl species; and E/Z isomerization of alkenes. The consequences of reactivity for biological activity and biophysical properties are also discussed.

Isolation of recombinant apolipoprotein E4 N-terminal domain by foam fractionation

Apolipoprotein (apo) E functions in lipoprotein metabolism as a low density lipoprotein receptor ligand. ApoE is comprised of two structural domains, a 22 kDa N-terminal (NT) domain that adopts a helix bundle conformation and a 10 kDa C-terminal domain with strong lipid binding affinity. The NT domain is capable of transforming aqueous phospholipid dispersions into discoidal reconstituted high density lipoprotein (rHDL) particles. Given the utility of apoE-NT as a structural component of rHDL, expression studies were conducted. A plasmid construct encoding a pelB leader sequence fused to the N-terminus of human apoE4 (residues 1–183) was transformed into Escherichia coli. Upon expression, the fusion protein is directed to the periplasmic space where leader peptidase cleaves the pelB sequence, generating mature apoE4-NT. In shaker flask expression cultures, apoE4-NT escapes the bacteria and accumulates in the medium. In a bioreactor setting, however, apoE4-NT was found to combine with gas and liquid components in the culture medium to generate large quantities of foam. When this foam was collected in an external vessel and collapsed into a liquid foamate, analysis revealed that apoE4-NT was the sole major protein present. The product protein was further isolated by heparin affinity chromatography (60–80 mg/liter bacterial culture), shown to be active in rHDL formulation, and documented to serve as an acceptor of effluxed cellular cholesterol. Thus, foam fractionation provides a streamlined process to produce recombinant apoE4-NT for biotechnology applications.

Studies of some bio-inspired liquid crystals

ABSTRACTThe main phase transition of lipid bilayers corresponds to a defined change from an ordered (gel) to a disordered (fluid liquid crystal) state of hydrocarbon chains, occurring in general at a defined temperature Tm. However, aqueous dispersions of pure anionic phospholipids, and particularly DMPG, may exhibit a ‘melting regime’, from Tmon until Tmoff, over ~10°C. The melting process depends on pH, ionic strength and several details of sample preparation. This paper makes a review on seven papers of the author with different collaborations, with the original proposal of pores in the DMPG melting transition. The focus is on structural results obtained from X-ray scattering, varying concentration and temperature, but integrating with results from other experimental techniques. Initially the effect of salt addition was separated from the effect of DMPG concentration. At DMPG concentrations higher than 70 mM a lamellar phase starts and a detailed study of the temperature variation of 150 mM DMPG allowed arrival to detailed pore model, focused in the surface fractions of pores in the bilayers. The model admits large and small pores in the melting regime and agrees with the new Lamellar Phase with pores (Lp) starting at T3. The large biological relevance is stressed.

Improving norbixin dispersibility and stability by liposomal encapsulation using the pH‐driven method

Norbixin, a carotenoid extracted from annatto seeds, is widely utilized as a natural pigment in foods, cosmetics and medicines. Its water solubility is relatively high under neutral or alkaline conditions but low under acidic conditions, which limits its application in some food products. This problem was overcome by utilizing liposomes to encapsulate the carotenoids so that they could be easily dispersed within acidic solutions. The norbixin was loaded into the liposomes using the pH-driven method. Liposomes were produced by passing aqueous phospholipid dispersions through a microfluidizer under high pressure. Norbixin was then added to the liposome dispersions at pH 7.0 and then driven into the hydrophobic domains of the phospholipid bilayers by acidifying the system. Measurements of the encapsulation efficiency showed that the norbixin was successfully loaded into the liposomes using the pH-driven method. X-ray diffraction analysis showed that the norbixin was in an amorphous state after incorporation into the liposomes. Encapsulation of norbixin within the liposomes was also shown to increase its water dispersibility and chemical stability under acidic pH conditions. The pH-driven method therefore provides a useful means of increasing the application of this bioactive carotenoid within functional foods and other products. © 2021 Society of Chemical Industry.

Determination of the efficient enzyme concentration for lytic digestion of vegetative cells but not spores in Schizosaccharomyces pombe

Received: 2016-05-24 | Accepted: 2016-05-30 | Available online: 2017-06-20 http://dx.doi.org/10.15414/afz.2017.20.01.20-22 The fact that lytic enzymes such as b-glucuronidase are capable of cell wall lysis, while the integrity of the spore is not affected, is used in the spore viability tests to investigate meiotic processes. Meiosis as a fundamental feature in all living organisms comprises of a complex tightly linked and mutually dependent processes most of which are scientific targets of many research institutions. The fission yeast Shizosaccharomyces pombe is a powerful tool for studies on eukaryotic meiosis. Mating of yeast strains of opposite mating types on nitrogen free medium results in spores production. Whereas not all cells undergo meiosis, some cells persist in vegetative stage even in the absence of nitrogen, this leads to generation of a mixed suspension of vegetative cells and spores. Thus, in order to separate spores from vegetative cells obtained mixture was exposed to lytic enzyme action. This treatment kills vegetative cells without affecting spores. To obtain the best and reproducible results of spore recovery and viability, different lytic conditions were analysed. Obtained results show, that use of b-glucuronidase as lytic enzyme for random spore analyses in the fission yeast is dose and time dependent. Keywords: b-glucuronidase, cell cycle, spore viability, Schizosaccharomycesn pombe References BAHALUL, M., KANETI, G. and KASHI, Y. (2010) Ether–zymolyase ascospore isolation procedure: an efficient protocol for ascosppores isolation in Saccharomyces cerevisiae yeast. Yeast, vol.27, no.12, pp.999–1003. doi: http://dx.doi.org/10.1002/yea.1808 DAWES, I.W., HARDIE, I.D. (1974) Selective killing of vegetative cells in sporulated cultures by exposure to diethyl ether. Mol Gen Genet, vol.131, no. 4, pp. 281–289. doi: http://dx.doi.org/10.1007/BF00264859 DONOVAN, D.M., KERR, D.E. and WALL, R.J. (2005) Engineering disease resistant cattle. Transgenic Res, vol. 14, pp. 563–567. doi: http://dx.doi.org/10.1007/s11248-005-0670-8 KHARE, A. K., SINGH, B., and SINGH, J. (2011) A fast and inexpensive method for random spore analysis in Schizosaccharomyces pombe . Yeast, vol. 28, pp. 527–533. doi: http://dx.doi.org/10.1002/yea.1855 MORENO, S., KLAR, A. and NURSE, P. (1991) Molecular genetic analysis of the fission yeast Schizosaccharomyces pombe . Methods Enzymol, vol . 194, pp. 795–823. http://dx.doi.org/10.1016/0076-6879(91)94059-L PARENTI-CASTELLI G, et al. (1974) Effect of soluble and membrane proteins upon diethyl ether extraction of aqueous phospholipid dispersions. Lipids, vol . 9, pp.221–228. doi: http://dx.doi.org/10.1007/BF02532197 SALAZAR, O. and ASENJO, J.A. (2007) Enzymatic lysis of microbial cells Review Biotechnology Letters , vol. 29, no. 7, pp. 985-994. doi: http://dx.doi.org/10.1007/s10529-007-9345-2 SMITH, G.R. (2009) Genetic Analysis of Meiotic Recombination in Schizosaccharomyces pombe Methods Mol Biol ., vol. 557, pp. 65–76. doi: http://dx.doi.org/10.1007/978-1-59745-527-5_6 YANG, Y.G, et al. (2000) The application of a novel lytic system to the recovery of recombinant proteins in E. coli. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) Acta biochimica et biophysica Sinica, vol. 32, no. 3, pp. 211–216. ŽUKAITE, V. and BIZIULEVICIUS, G.A. (2000) Acceleration of hyaluronidase production in the course of batch cultivation of Clostridium perfringens can be achieved with bacteriolytic enzymes. Lett Appl Microbiol , vol. 30, pp. 203–206. doi: http://dx.doi.org/10.1046/j.1472-765x.2000.00693.x

Enabling Marangoni flow at air-liquid interfaces through deposition of aerosolized lipid dispersions

It has long been known that deposited drops of surfactant solution induce Marangoni flows at air-liquid interfaces. These surfactant drops create a surface tension gradient, which causes an outward flow at the fluid interface. We show that aqueous phospholipid dispersions may be used for this same purpose. In aqueous dispersions, phospholipids aggregate into vesicles that are not surface-active; therefore, drops of these dispersions do not initiate Marangoni flow. However, aerosolization of these dispersions disrupts the vesicles, allowing access to the surface-active monomers within. These lipid monomers do have the ability to induce Marangoni flow. We hypothesize that monomers released from broken vesicles adsorb on the surfaces of individual aerosol droplets and then create localized surface tension reduction upon droplet deposition. Deposition of lipid monomers via aerosolization produces surface tensions as low as 1mN/m on water. In addition, aerosolized lipid deposition also drives Marangoni flow on entangled polymer solution subphases with low initial surface tensions (∼34mN/m). The fact that aerosolization of phospholipids naturally found within pulmonary surfactant can drive Marangoni flows on low surface tension liquids suggests that aerosolized lipids may be used to promote uniform pulmonary drug delivery without the need for exogenous spreading agents.

Collapse of a lipid-coated nanobubble and subsequent liposome formation.

We investigate the collapse of a lipid-coated nanobubble and subsequent formation of a lipid vesicle by coarse grained molecular dynamics simulations. A spherical nanobubble coated with a phospholipid monolayer in water is a model of an aqueous dispersion of phospholipids under negative pressure during sonication. When subjected to a positive pressure, the bubble shape deforms into an irregular spherical shape and the monolayer starts to buckle and fold locally. The local folds grow rapidly in multiple directions and forming a discoidal membrane with folds of various amplitudes. Folds of small amplitude disappear in due course and the membrane develops into a unilamellar vesicle via a bowl shape. Folds with large amplitude develop into a bowl shape and a multivesicular shape forms. The membrane shape due to bubble collapse can be an important factor governing the vesicular shape during sonication.

0313 脂質殻ナノ気泡の分子シミュレーション

We investigate the collapsing mechanisms of a lipid coated nanobubble and the subsequent lipid vesicle formation by coarse grained molecular dynamics (CGMD) simulations. A preformed nanobubble coated by lipid monolayer in water is a model of an aqueous dispersion of phospholipids under negative pressure in sonication. Relaxing to a positive pressure, the spherical bubble shape deforms into irregular prolate ellipsoidal shape and the monolayer starts to fold from an apsis of the ellipsoid. The local folding is rapidly propagated in the ellipsoid, pushing gas core, and a discoidal membrane forms. The discoidal membrane develops into a unilamellar vesicle via bowl shape.

The influence of hydrophilic spacers on the phase behavior of ether lipids

Oxyethylene (EO) units known from nonionic amphiphiles of the type C n (EO) m have been used as a spacer between the hydrophobic and hydrophilic parts of phospholipid molecules. Here we describe the synthesis of the compounds and present results of aqueous dispersions of phospholipids with 3 and 6 EO units. The mechanism of the phase transition from ordered gel to a liquid-crystalline L α-phase depends on the number of the EO units. In the case of DH(EO) 3PC, the lipid head groups are interdigitated in the L β-phase. On heating, first the head groups de-interdigitate and the melting of the hydrocarbon chains starts only after reaching a stretched head group conformation. While on heating the lamellar repeat distance changes continuously, the cooling process is more complex showing two regimes for the decrease of the lamellar repeat distance. In the case of DH(EO) 6PC, the same transition occurs in a single step without any hysteresis between heating and cooling.

ANTIOXIDANT ACTIVITIES OF VITAMINS E AND C IN A NOVEL LIPOSOME SYSTEM

ABSTRACT A new model liposome made of anionic and cationic surfactants and methyl linoleate was used to investigate the antioxidant activity of vitamins E and C. The spontaneous vesicle formation was demonstrated by transmission electron microscopy with a negative-staining technique. The model liposome was kept in the dark at a temperature of 310K and the progress of the methyl linoleate oxidation was evaluated by measurement of conjugated dienes hydroperoxides and malondialdehydes. The results indicate that vitamins E and C could inhibit the lipid oxidation as a chain-breaking antioxidant, respectively. Further, the combination of vitamins E and C causes an antioxidant synergistic effect in the model system. It is shown that the model liposome can be suitable for simulating biomembranes as a convenient and useful alternative model system. PRACTICAL APPLICATIONS Phospholipid vesicles, which are produced from the aqueous dispersion of single-component phospholipids, when formed in vitro are usual models of biomembranes for investigation of the antioxidant activities in biological systems. However, such lipid vesicles are not the equilibrium aggregation state and can be considered as metastable structures of high kinetic stability but thermodynamically nonstable. The present study shows that the lipid vesicles formed in an aqueous mixture of cetyltrimethylammonium bromide, sodium dodecyl sulfonate and methyl linoleate by using sonication could be used as appropriate models for in vitro mimicking the behavior of biological membranes and those oxidation processes occurring within them. This model liposome is a stable and convenient alternative model system. Furthermore, the comparison of the antioxidant effects of vitamins E and C in this study may be helpful to understand the mechanism of antioxidant action in biological system.

Import and fate of fluorescent analogs of oxidized phospholipids in vascular smooth muscle cells

Lipid oxidation is now thought to be an initiating and sustaining event in atherogenesis. Oxidatively fragmented phospholipids, namely 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), present in minimally modified LDL and atherosclerotic lesions, have been reported to elicit a wide range of pathophysiological responses in the cells of the vascular wall. Nevertheless, the question of their potential sites of action and their primary molecular targets remains open. To address this issue, a series of fluorescently labeled analogs, which differ with regard to structure and binding site of the fluorophore, were synthesized and used as tools for studying the uptake, intracellular stability, and distribution of PGPC and POVPC in vascular smooth muscle cells (VSMCs). We demonstrate that in accordance with their lysophospholipid-like structure, these highly similar molecules transferred rapidly either from aqueous phospholipid dispersions or preloaded native LDL into VSMCs, producing disparate fluorescence patterns irrespective of the attached fluorophore. PGPC derivatives were translocated to the lysosomes. In sharp contrast, POVPC analogs were initially captured in the plasma membrane, most likely in consequence of the formation of covalent adducts with free amino and sulfhydryl groups of proteins and phospholipids. LDL internalization is not required for cellular lipid uptake. Collectively, our data provide evidence that oxidized phospholipids, owing to their high exchangeability between lipoproteins and cell membranes, may act within a short time on different cellular sites in VSMCs and affect various lipid and protein components through physical or chemical interactions, which might then serve as starting points for intracellular signaling.

GP005 Identification of the Streptococcus pyogenes fas two-component signal transduction system-controlled regulon using differential proteome analysis

Physical properties of Escherichia coli membrane lipids in logarithmic- and stationary-phase cells were studied by measuring the fluorescence polarization change of cis- and trans-parinaric acid as a function of temperature. In aqueous dispersions of phospholipids extracted from cytoplasmic and outer membranes of cells of differing growth phase, a similar polarization increase was observed over the range from physiological temperature to below 0°C, and nearly the same transition ratios were obtained in all samples. The cytoplasmic membrane of both of the growth-phase cells showed a higher polarization ratio above the transition temperatures, compared to that in the aqueous dispersion of phospholipids. The polarization ratios below the transition temperatures of these specimens were lower than the value obtained with the lipids, especially in the stationary-phase specimens. The outer membrane specimens showed a similar polarization change but the transition temperature ranges were considerably higher both in the logarithmic- and the stationary-phase specimens, compared to those in the cytoplasmic membrane specimens. Freeze-thawing of logarithmic-phase cells showed the emergence of activity of certain enzymes which are known to be located in the membranes. The stationary-phase cells did not suffer from any such deleterious effect and maintained a high level of cell viability in a similar treatment. These results indicate that in the stationary-phase cell membranes lipids are in a highly ordered state, and the lipid state causes a membrane stability which results in the high resistance of the cell to freeze-thawing.

Odorants generated by thermally induced degradation of phospholipids.

The qualitative and quantitative aroma composition of heated aqueous dispersions of egg phosphatidylcholine (PC) and egg phosphatidylethanolamine (PE) were characterized by aroma extract dilution analysis and isotope dilution assay. On the basis of FD-factors and odor activity values, trans-4,5-epoxy-(E)-2-decenal was found to be the most potent odorant followed by (E,E)-2,4-decadienal, 1-octen-3-one, and hexanal. The amount of (E,E)-2,4-decadienal in PC was about 20-fold higher compared to PE, while hexanal was the major odor-active compound in the PE sample. (E,Z,Z)-2,4,7-Tridecatrienal was identified for the first time as an odor-active volatile constituent of heated phospholipids exhibiting a characteristic egg white-like note. Further odorants first reported in thermally treated phospholipids were (Z)-2-decenal, (E)-2-decenal, and (E)-2-undecenal. Differences in the fatty acid composition of PC and PE can only partially explain the quantitative results found in this study, thus suggesting that further parameters may influence the formation of carbonyls from heated aqueous dispersions of phospholipids.

Aggregate Structure in Dilute Aqueous Dispersions of Phospholipids, Fatty Acids, and Lysophospholipids

Cryo-transmission electron microscopy (cryo-TEM) was employed to investigate the aggregate structure in dilute aqueous dispersions of egg-phosphatidylcholine (EPC), oleic acid (OA), and the lysophospholipid monooleoylphosphatidylcholine (MOPC). At physiological pH and salt concentration, a relatively monodisperse population of unilamellar liposomes was detected in mixtures containing equimolar concentrations of the three components. Threadlike micelles constituted the dominant aggregate structure in samples containing high concentrations of MOPC. Excess fatty acid forced, on the other hand, the system toward structures with net negative curvature. In the absence of phospholipid, cryo-TEM revealed bilayer fragments in coexistence with threadlike micelles in mixtures containing the same molar amount of MOPC and OA. External addition of MOPC to preformed EPC liposomes gave rise to a concentration dependent evolution of intermediate structures, including open liposomes and bilayer fragments. The structural re...

A Fourier transform infrared spectroscopic study of the interaction of alkaline earth cations with the negatively charged phospholipid 1,2-dimyristoyl- sn-glycero-3-phosphoglycerol

The interaction of aqueous phospholipid dispersions of negatively charged 1,2-dimyristoyl- sn-glycero-3-phosphoglycerol, sodium salt (DMPG) with the divalent cations Mg 2+, Ca 2+ and Sr 2+ at equimolar ratios in 100 mM NaCl at pH 7 was investigated by Fourier transform infrared spectroscopy. The binding of the three cations induces a crystalline-like gel phase with highly ordered and rigid all- trans acyl chains. These features are observed after storage below room temperature for 24 h. When the gel phase is heated after prolonged incubation at low temperature phase transitions into the liquid crystalline phase are observed at 58°C for the DMPG:Sr 2+, 65°C for the DMPG:Mg 2+, and 80°C for the DMPG:Ca 2+ complex. By subsequent cooling from temperatures above T m these complexes retain the features of a liquid crystalline phase with disordered acyl chains until a metastable gel phase is formed at temperatures between 38 and 32°C. This phase is characterized by predominantly all- trans acyl chains, arranged in a loosely packed hexagonal or distorted hexagonal subcell lattice. Reheating the DMPG:Sr 2+ samples after a storage time of 2 h at 4°C results in the transition of the metastable gel to the liquid crystalline phase at 35°C. This phase transition into the liquid crystalline state at 35°C is also observed for the Mg 2+ complex. However, for DMPG:Mg 2+ at higher temperatures, a partial recrystallization of the acyl chains occurs and the high temperature phase transition at 65°C is also detected. In contrast, DMPG:Ca 2+ exhibits only the phase transition at 80°C from the crystalline gel into the fluid state upon reheating. Below 20°C, the rate of conversion from the metastable gel to a thermodynamically stable, crystalline-like gel phase decreases in the order Ca 2+≫Mg 2+>Sr 2+. This conversion into the crystalline gel phase is accompanied by a complete dehydration of the phosphate groups in DMPG:Mg 2+ and by a reorientation of the polar lipid head groups in DMPG:Ca 2+ and in DMPG:Sr 2+. The primary binding sites of the cations are the PO 2 − groups of the phosphodiester moiety. Our infrared spectroscopic results suggest a deep penetration of the divalent cations into the polar head group region of DMPG bilayers, whereby the ester carbonyl groups, located in the interfacial region of the bilayers, are indirectly affected by strong hydrogen bonding of immobilized water molecules. In the liquid crystalline phase, the interaction of all three cations with DMPG is weak, but still observable in the infrared spectra of the DMPG:Ca 2+ complex by a slight ordering effect induced in the acyl chains, when compared to pure DMPG liposomes.

LIPOSOME MICROENCAPSULATIONS WITHOUT USING ANY ORGANIC SOLVENT

ABSTRACT Liposomes are microscopic vesicles composed of phospholipid bilayers surrounding discrete aqueous compartment. Eventhough phospholipid bilayers are formed spontaneously in water, their conventional preparation methods involve either organic solvents or detergent molecules. Organic solvents may create a serious safety problem during liposome manufacturing especially on a large scale and can cause denaturation of proteins and affect the membrane properties. Liposomes are very promising carriers for protein drugs which have aroused great interests in the field of pharmaceutics in these recent years. The objective of this study was to develop a new dispersion process to prepare liposomes without the usage of organic solvents and detergents. The approach involved the usage of a microfludizer for the extrusion and homogenization of aqueous dispersion of phospholipids, and a modified evaporator in order to increase lipid thin film surface area. The properties of the liposomes prepared by the novel method were superior to the liposomes prepared by conventional methods.

Self-Assembly of Styrylthiophene Amphiphiles in Aqueous Dispersions and Interfacial Films: Aggregate Structure, Assembly Properties, Photochemistry, and Photophysics

Fatty acids and phosphatidyl choline derivatives incorporating the photoreactive trans-styrylthiophene chromophore have been prepared and studied in Langmuir−Blodgett films and aqueous dispersions, respectively. Both absorption and fluorescence spectra in the assemblies show prominent shifts indicative of aggregation similar to that observed with similar trans-stilbene and trans-azobenzene derivatives previously investigated. Studies of aqueous dispersions of the pure styrylthiophene phospholipids indicate the formation of structures much larger than the typical small unilamellar bilayer vesicles formed from saturated phospholipids of comparable chain length. Studies of the disaggregation process for two of the phospholipids give aggregation numbers of approximately 2 and 6, corresponding to 4 and 12 styrylthiophene units per aggregate. The stability of these aggregates is very similar to those of corresponding stilbene aggregates, and on the basis of spectral similarities it seems reasonable to propose a “pinwheel” fraction of a glide or herringbone structure as the “unit aggregate” and predominate species present in the aqueous dispersions. However, the chief photoreaction observed for the aqueous dispersions of the phospholipids is dimerization to form the syn head-to-head dimer, consistent with topologically controlled reaction from a translation structure in which nearest-neighbor chromophores are aligned parallel. Simulations suggest that while a translation layer structure may be of lowest energy, glide layer structures that show good agreement between measured and predicted properties are also energetically accessible. A possible explanation for the observed reactivity and photophysics is that within the aqueous dispersions there may be equilibration between “glide” and “translation” structures. When mixtures of the styrylthiophene phospholipids and saturated phospholipids are codispersed, bilayer vesicles are formed that are capable of entrapping reagents such as carboxyfluorescein. Irradiation of the aggregated trans-styrylthiophene in these vesicles leads to release of the entrapped carboxyfluorescein concurrent with photodimerization of the styrylthiophene. The pattern of reagent release is similar to that observed upon photoisomerization of the corresponding azobenzene aggregates and suggests a “catastrophic” destruction of the vesicles.

Supramolecular Aggregates of Azobenzene Phospholipids and Related Compounds in Bilayer Assemblies and Other Microheterogeneous Media: Structure, Properties, and Photoreactivity1

A number of phosphatidyl choline derivatives containing trans-azobenzene units in the fatty ester backbone have been synthesized and studied in aqueous dispersions both pure and in the presence of saturated and unsaturated phospholipids. The structures of the assemblies formed have been investigated by microcalorimetry, dynamic light scattering, cryo-transmission electron microscopy, and reagent entrapment. While many of the mixed phospholipid dispersions give evidence for the formation of small unilamellar vesicles, the aqueous dispersions of pure azobenzene phospholipids (APL's) give evidence for several different structures, including relatively large plates in at least one case. The azobenzenes show strong evidence of “H” aggregate formation both in the pure and mixed dispersions. The aggregation number has been estimated for several of the APL's and found to be typically 3 or a multiple thereof. On the basis of simulations and studies with similar stilbene phospholipids as well as on the strong induced circular dichroism signals observed for the aggregate, we infer a chiral “pinwheel” unit aggregate structure similar to that found for several aromatics. The azobenzenes in the aqueous dispersions have been found to photoisomerize to give cis-rich photostationary states; the cis-azobenzenes show no evidence for aggregation and no induced circular dichroism. The cis-azobenzenes can be isomerized back to the trans either by irradiation or by thermal paths. Mixed aqueous dispersions of trans-APL's with saturated or unsaturated phospholipids can be prepared which entrap the fluorescent dye carboxyfluorescein (CF) under conditions where the CF fluorescence is very low due to self-quenching. By varying the APL/host phospholipid ratio the azobenzene can be aggregate, monomer, or dimer. In cases where the azobenzene is monomer or dimer, irradiation produces complete isomerization but little “leakage” of CF from the vesicle interior. In contrast, where the azobenzene is predominantly aggregate, irradiation results in both photoisomerization and reagent release. That photoisomerization in the latter case can result in “catastrophic” destruction of the vesicle can also be shown by cryo-transmission electron microscopy.

Probing the critical unilamellar state of membranes.

Aqueous dispersions of membrane phospholipids comprised of multilamellar vesicles (MLVs) will spontaneously transform to a stable unilamellar structure equivalent to the membrane bilayer, but only at a critical temperature T*. Since much of the evidence for this transformation derives from equilibrium thermodynamic studies, a description of the molecular and topological events occurring as the critical unilamellar state assembles has not previously been possible. Here we report experiments that provide evidence of a spontaneous topological change from MLVs towards unilamellar vesicles (ULVs) at T*. By applying a shearing stress to vesicle suspensions we have observed a decrease of approximately 25% in the force required to cause bilayers to leak; this decrease is confined to temperatures near T*. The T* values observed agree with those previously obtained by equilibrium methods. Using the method with total lipid extracts from normal biological membranes confirms that T* equals the physiological temperature of the original membranes.

Evaluating spin diffusion in MAS-NOESY spectra of phospholipid multibilayers

Biological semisolids such as aqueous phospholipid dispersions are amenable to experiments that combine magic-angle spinning (MAS) to yield high-resolution 1H NMR spectra and two-dimensional nuclear Overhauser spectroscopy (NOESY) to estimate proton-proton distances. Using several selectively deuterated dimyristoylphosphatidylcholines, a systematic investigation has been made into the origin of an anomalous NOESY crosspeak between methyl groups of the lipid headgroup and the acyl chains. Although the spin-relaxation and line-narrowing behavior in these multilamellar systems argue against efficient spin diffusion, interdigitated or chain-bendback structural models that bring these 1H nuclei into close proximity are not implicated by the MAS-NOESY results for this phospholipid system. A mechanism has been proposed for spin communication between the two types of methyl protons within a conventional lipid bilayer structure: through-space interactions link the headgroup and backbone protons on adjacent molecules, and then spin diffusion occurs along the acyl chains.