Phosphatidylcholine Large Unilamellar Vesicles Research Articles

Physicochemical properties of L-alpha dipalmitoyl phosphatidylcholine large unilamellar vesicles: Effect of hydrophobic block (PLA/PCL) of amphipathic diblock copolymers

In the present work, we show how amphipathic diblock copolymers affect the physicochemical properties of the lipid bilayer of DPPC liposome. Diblock copolymers proposed for this study are focused in the difference between PLA and PCL hydrophobic block, because PLA and PCL differ in their glass transition temperature, where a higher ratio of PLA, lowers the flexibility of the diblock copolymer. On the contrary, a greater proportion of PCL makes the diblock copolymer more flexible. This flexibility difference between hydrophobic block would affect the physicochemical properties of lipid bilayer of DPPC. The difference of rigidity or flexibility of hydrophobic block and their interaction with DPPC large unilamellar vesicles (LUVs) was evaluated at low and high copolymers concentration. The copolymer concentrations used were chosen based on their respective cmc. We measure (a) Thermotropic behavior from GP of Laurdan and fluorescence anisotropy of DPH; (b) Relation between wavelength excitation and generalized polarization of Laurdan; (c) Time-resolved fluorescence anisotropy of DPH; (d) Water outflow through the lipid bilayer and (e) calcein release from DPPC LUVs. Furthermore, large unilamellar vesicles in the absence and in the presence of different copolymers were characterized by size and zeta-potential. The results show that the diblock copolymer at high PLA/PCL ratio, that is, greater rigidity of hydrophobic block produces an increase of the phase transition temperature (Tm). For DPPC LUVs, Tm increase 3.5 °C at low and about 4.5 °C at high copolymers concentration, sensed by Laurdan and DPH fluorescent probes, although the DPPC/copolymers molar ratio for Cop4 is higher than Cop3, Cop2 and Cop1. In addition, we observed a decrease in the polarity of microenvironments in the bilayer and an increase in the order of the acyl chains in the bilayer to a high proportion of PLA. Furthermore, the presence of diblock copolymer with high proportion of PLA, decreases water outflow from DPPC liposome and water efflux is slower; leading to a decrease in calcein release from DPPC liposomes. Our results clearly show that the greater the stiffness of the hydrophobic block, greater degree of packaging of the lipid bilayer, greater the order of the acyl chains, and greater retention of water and calcein inside the liposome. Therefore, the presence of AB-type diblock copolymers with a more rigid hydrophobic block, stabilizes the lipid bilayer and would allow a more controlled release of water, and encapsulated molecules inside of the DPPC liposome.

Interaction between amphipathic triblock copolymers and L-α-dipalmitoyl phosphatidylcholine large unilamellar vesicles

This study contributes to an understanding of how different polymeric structures, in special triblock copolymers can interact with the lipid bilayer. To study the phospholipid-copolymer vesicles system, we report the effect of two amphipathic triblock copolymers of the type BAB, i.e., hydrophobic-hydrophilic-hydrophobic triblock copolymers arranged as poly(ε-caprolactone)-poly(ethylene oxide)-poly(ε-caprolactone) (PCLn-PEOm-PCLn), where n=12 and m=45 for COP1 and n=16 and m=104 for COP2, on the dynamic and structural properties of dipalmitoyl-phosphatidylcholine (DPPC) large unilamellar vesicles (LUVs). The interaction between the copolymers and DPPC LUVs was evaluated by means of several techniques: (a) Photographs of the dispersion for evaluation of colloidal stability; (b) Thermotropic behavior from generalized polarization of Laurdan and fluorescence anisotropy of DPH (c) Main phase transition temperature determination; (d) Order parameters and limiting anisotropy by time-resolved fluorescence anisotropy measurements; (e) Water outflow through the lipid bilayer and (f) Calcein release from DPPC LUVs. Steady-state fluorescence measurements as a function of temperature show a typical behavior. Laurdan and DPH are fluorescent probes that sense the interface and the inner part of the bilayer, respectively. Both copolymers increase the Tm value of DPPC LUVs sensed by DPH, i.e., in the inner part of the bilayer. On the contrary, only COP2 had an effect on increasing the Tm value at the interface of the bilayer. At low temperature, in the gel phase, the presence of the copolymers produced a slight decrease in generalized polarization of Laurdan sensed in the interface of the lipid bilayer, but in the liquid-crystalline phase it produced an increase. In contrast, the order parameters obtained from time-resolved fluorescence anisotropy of DPH show an increase in the presence of the copolymers in the gel phase, but a decrease in the liquid-crystalline phase. COP2 produces a greater effect than COP1 in decreasing the water outflow through DPPC LUVs in the same concentration range. Furthermore, calcein release was decreased to a minimum at low copolymer concentration; however, at high concentration, release factor percentage (RF%) increased slightly without reaching the values obtained in the absence of copolymers. Therefore, the copolymer concentrations studied decrease the calcein release from the liposome.

Phosphatidylserine, a Lipid Present at the Outer Membrane Leaflet of Cancer Cells, Hinders the Insertion of pHLIP, a Potential Cancer Cell Marker

The pH-Low Insertion Peptide (pHLIP) has shown the ability to insert into membranes when acidic conditions are present. pHLIP can be found in three different states: a random coil in pH 8 solution devoid of lipids (state I), a surface bound form when lipids are present (state II), and a transmembrane helix in acidic conditions (state III). From this ability of insertion based on pH, pHLIP has been shown to effectively target diseases in which the extracellular pH of the tissue has become acidic. Cancer is an example of such a disease, and it has been shown that pHLIP can target cancerous cells. One concern with this targeting is that some cancerous cells are known to display phosphatidylserine (PS) in the outer leaflet of the their plasma membranes, as opposed to normal cells. However, little is known about how pHLIP's tumor targeting is influenced by this increased PS content. Here, we study how pHLIP properties are influenced by the presence of PS by using phosphatidylcholine (PC) large unilamellar vesicles (LUV's) that contain PS. Circular dichroism data shows that the characteristic three states are maintained in the presence of PS. Our results show that pHLIP has higher affinity to PC than to PS. We also found that when LUV's contain PS, the insertion pKa decreases compared to LUV's only containing PC with a midpoint at 0.9±0.4 percent. This decrease in insertion pKa might limit pHLIP's targeting of mildly acidic tumors. We added sodium chloride to screen any electrostatic repulsion; however, no change in the pKa was observed. This suggests that the effect of PS on the binding and insertion of pHLIP is not merely due to electrostatic repulsion.

Rapid transmembrane diffusion of ceramide and dihydroceramide spin-labelled analogues in the liquid ordered phase

In order to study the basic physical phenomena underlying complex lipid transbilayer movement in biological membranes, we have measured the transmembrane diffusion of spin-labelled analogues of sphingolipids in phosphatidylcholine (PC) large unilamellar vesicles in the absence or presence of cholesterol, going from a fluid ( liquid disordered) l(d), phase to a more viscous, liquid ordered (l(o)), phase. We have found cholesterol to reduce the transverse diffusion of glucosylceramide (GlcCer) and galactosylceramide (GalCer) in a concentration-dependent manner. However, surprisingly, we could neither detect any influence of cholesterol on the rapid flip-flop of ceramide nor on the flip-flop of dihydroceramide, for which the tau(1/2) of flip-flop remains in the order of 1 minute at 20 degrees C in the presence of cholesterol. As a consequence of rapid flip-flop of ceramide in both the l(o) and the l(d) phase, ceramide is likely to distribute between the two monolayers of a membrane, and could in principle partition into segregated domains in each side of the plasma membrane of eukaryotic cells.

Disk-like Complex Formation by Apolipoprotein-Lipid Membrane Interaction and Relevance to High-Density Lipoprotein Formation

The interaction of apolipoprotein A-I (apoA-I) with transmembrane ATP-binding cassette transporter A1 (ABCA1) is a crucial step for high-density lipoprotein (HDL) formation, however, its molecular mechanism is less well understood. Here, we used apoA-I and its model peptide, Ac-18A-NH(2), to investigate their interaction with mixed membranes of phosphatidylcholine (PC) with phosphatidylethanolamine (PE) or sphingomyelin (SM). It was shown that PE, possessing the negative spontaneous curvature, increased both the degree of hydration at the membrane interface and the acyl chain order, and that Ac-18A-NH(2) had opposite effects to PE, since the alpha-helix formation at the membrane surface induced positive curvature strain. In addition, increased PE in PC/PE large unilamellar vesicles (LUVs) enhanced the peptide's binding. Although SM significantly lowered the peptide binding capacity, the peptide's binding to PC/SM LUV led to membrane disruption. The interaction with PC/SM LUVs was then investigated using ApoA-I. The spontaneous rHDL formation from pure PC LUV proceeded very slowly at 37 degrees C, but SM-rich PC/SM LUVs, which are in a gel/liquid-disordered (L(d)) phase at this temperature, were rapidly solubilized to form rHDL by apoA-I. The addition of cholesterol decreased the rate of the rHDL formation and induced the selective extraction of lipids by apoA-I, which preferably extracted lipids of L(d) phase rather than lipids of liquid-ordered (L(o)) phase. These results suggest that heterogeneous interface of the mixed membranes facilitates the insertion of apoA-I and induces L(d) phase-selective lipid extraction to form rHDL, and are compatible with recent cell works on the apoA-I-dependent HDL generation.

Spontaneous reconstitution of discoidal HDL from sphingomyelin-containing model membranes by apolipoprotein A-I

Nascent HDL is known to be formed by the interaction of apolipoprotein A-I (apoA-I) with transmembrane ABCA1, but the molecular mechanism by which nascent HDL forms is less well understood. Here, we studied how reconstituted high density lipoprotein (rHDL) forms spontaneously on the interaction of apoA-I with model membranes. The formation of rHDL from pure phosphatidylcholine (PC) large unilamellar vesicles (LUVs) proceeded very slowly at 37.0 degrees C, but sphingomyelin (SM) -rich PC/SM LUVs, which are in a gel/liquid-disordered phase (L(d) phase) at this temperature, were rapidly microsolubilized to form rHDL by apoA-I. The addition of cholesterol decreased the rate at which rHDL formed and induced the selective extraction of lipids by apoA-I, which preferably extracted lipids of L(d) phase rather than lipids of liquid-ordered phase. In addition, apoA-I extracted lipids from the outer and inner leaflets of LUVs simultaneously. These results suggest that the heterogeneous interface of the mixed membranes facilitates the insertion of apoA-I and induces L(d) phase-selective but leaflet-nonselective lipid extraction to form rHDL; they are compatible with recent cell works on apoA-I-dependent HDL generation.

Cholesterol Depletion and Genistein as Tools to Promote F508delCFTR Retention at the Plasma Membrane

Background/aims: F508delCFTR-, but not wtCFTR-, expressing fibroblasts resemble Niemann Pick type C cells in the massive intracellular accumulation of free cholesterol. The recruitment and activation of F508delCFTR by cholesterol depletion was studied. Methods: Filipin staining, forskolin-stimulated anion efflux and FITC-dextran uptake were studied in control cells and fibroblasts treated with 2-hydroxypropyl β-cyclodextrin phosphatidylcholine large unilamellar vesicles to deplete cellular free cholesterol.Results: Treatment of F508delCFTR-, but not wtCFTR-, expressing fibroblasts with 2-hydroxypropyl β-cyclodextrin resulted in a reduction in cellular cholesterol and a potentiation of the forskolin-induced anion efflux. In addition, forskolin also promoted a massive increase in the rate of endocytosis in F508delCFTR fibroblasts, which was absent in genistein- or cyclodextrin-treated cultures.Conclusion: The results not only suggest that reducing cellular cholesterol may serve as pharmacotherapeutic tool in the treatment of cystic fibrosis but also reveal a novel mechanism for genistein regulation of F508delCFTR, i.e. retention by inhibition of endocytosis.

Solvent and pH Effects on the Antioxidant Activity of Caffeic and Other Phenolic Acids

The antioxidant activity of several phenolic acids and esters has been investigated both in organic solutions and in large unilamellar phosphatidylcholine vesicles. In solution these compounds behaved as good antioxidants, with the exception of protocatechuic acid, due to the presence of the catechol moiety. Because their antioxidant activity followed an inverse dependence on the magnitude of their O-H bond dissociation enthalpies (BDE), the key mechanism of the chain-breaking action was attributed to hydrogen atom transfer (HAT) from the phenolic OH to peroxyl radicals. In unilamellar vesicles the antioxidant activity was strongly dependent on the pH of the buffer solution. In acid media (pH 4) all of the examined phenolic acids or esters behaved as weak inhibitors of peroxidation, whereas, with increasing pH, their antioxidant activity increased substantially, becoming comparable to or even better than that of Trolox. At pH 8 they also gave rise to lag phases 2-3 times longer than that of Trolox. The increased activity being observed in proximity of the pK(a) value corresponding to the ionization of one of the catecholic hydroxyl groups, this effect has been attributed to the high antioxidant activity of the phenolate anion.

Controlling Association of Vesicle Embedded Peptides by Alteration of the Physical State of the Lipid Matrix

We report here the reversible association of a designed peptide embedded in a lipid membrane through a stimulus-sensitive trigger that changes the physical state of the bilayer matrix. A peptide designed with the classical 4-3 heptad repeat of coiled coils, equipped with leucine residues at all canonical interface positions, TH1, was rendered membrane soluble by replacement of all exterior residues with randomly selected hydrophobic amino acids. Insertion of TH1 into large unilamellar phosphatidylcholine vesicles was followed by monitoring tryptophan fluorescence. Peptide insertion was observed when the lipids were in the liquid-crystalline state [1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)] but not when they were in the crystalline phase [1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)]. Formation of a trimeric alpha-helical bundle in lipid bilayers was followed by fluorescence resonance energy transfer. Global fit analysis revealed a monomer--trimer equilibrium with a dissociation constant of around 10(-5) [corrected] MF(2). A lipid mixture composed of DPPC and POPC exhibiting a phase transition at 34 degrees C between a crystalline/liquid-crystalline coexistence region and a completely miscible liquid-crystalline phase was used to control the formation of the trimeric peptide bundle. TH1 is phase excluded in crystalline DPPC domains below 34 degrees C, leading to a larger number of trimers. However, when the DPPC domains are dispersed at temperatures above 34 degrees C, the number of trimers is reduced.

Stabilization of phosphatidylcholine coatings in capillary electrophoresis by increase in membrane rigidity

Divalent cations affect the stability and structure of phospholipid vesicles and also the binding and immobilization of proteins into phospholipid membranes. The effect of calcium, magnesium, and zinc on zwitterionic phosphatidylcholine (PC) coatings in fused silica capillaries for electrophoresis was the primary interest in this work. In addition, the effect of temperature on the coating stability was investigated by coating 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes at temperatures above and below the gel- to fluid-state transition. All coatings were performed with PC large unilamellar vesicles (LUV) in 40 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) at pH 7.4 as basic solution. HEPES (40 mM) at pH 7.4 was used as background electrolyte (BGE) throughout the study. The stability of the coating was studied by measuring the electroosmotic flow. A molar ratio of 1:3 PC/Ca2+ or PC/Mg2+ gave the best coating stability owing to the increased rigidity of the phospholipid membrane furnished by the divalent metal ions. Better results were obtained with DPPC in the more rigid gel state than in the fluid state: the electroosmotic flow was much suppressed and the PC coating was stabilized. Coating the fused silica capillary with PC liposome–metal ion buffer solutions resulted in good electrophoretic separation of basic model proteins (pI-values 7.8–11.0). The electrophoretic results demonstrate the importance of stabilizing the phospholipid coating on fused silica capillaries, either by the addition of divalent metal ions (Ca2+, Mg2+, or Zn2+) or by working in the gel-state region of the phospholipid.

Stabilization of phosphatidylcholine coatings in capillary electrophoresis by increase in membrane rigidity

Divalent cations affect the stability and structure of phospholipid vesicles and also the binding and immobilization of proteins into phospholipid membranes. The effect of calcium, magnesium, and zinc on zwitterionic phosphatidylcholine (PC) coatings in fused silica capillaries for electrophoresis was the primary interest in this work. In addition, the effect of temperature on the coating stability was investigated by coating 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) liposomes at temperatures above and below the gel- to fluid-state transition. All coatings were performed with PC large unilamellar vesicles (LUV) in 40 mM N-(2-hydroxyethyl)piperazine- N′-(2-ethanesulfonic acid) (HEPES) at pH 7.4 as basic solution. HEPES (40 mM) at pH 7.4 was used as background electrolyte (BGE) throughout the study. The stability of the coating was studied by measuring the electroosmotic flow. A molar ratio of 1:3 PC/Ca 2+ or PC/Mg 2+ gave the best coating stability owing to the increased rigidity of the phospholipid membrane furnished by the divalent metal ions. Better results were obtained with DPPC in the more rigid gel state than in the fluid state: the electroosmotic flow was much suppressed and the PC coating was stabilized. Coating the fused silica capillary with PC liposome–metal ion buffer solutions resulted in good electrophoretic separation of basic model proteins (p I-values 7.8–11.0). The electrophoretic results demonstrate the importance of stabilizing the phospholipid coating on fused silica capillaries, either by the addition of divalent metal ions (Ca 2+, Mg 2+, or Zn 2+) or by working in the gel-state region of the phospholipid.

In Vitro Skin Permeation and Retention of Paromomycin from Liposomes for Topical Treatment of the Cutaneous Leishmaniasis

Paromomycin (PA), a very hydrophilic antibiotic, has been tested as an alternative topical treatment against cutaneous leishmaniasis (CL). Although this treatment has shown promising results, it has not been successful in accelerating the recovery in most cases. This could be attributed to the low skin penetration of PA. Liposomal formulations usually provide sustained and enhanced drug levels in skin. The aim of this study was to prepare liposomal formulations containing PA and to investigate their potential as topical delivery systems of this antileishmanial. Large multilamellar vesicles (MLVs) were prepared by conventional solvent evaporation method. Large unilamellar vesicles (LUVs) were prepared by reverse‐phase evaporation method. The lipids used were soybean phosphatidylcholine (PC) and PC:cholesterol (CH) (molar ratio 1:1). The skin permeation experiments across stripped and normal hairless mice skin were performed in modified Franz diffusion cells. The PA entrapment in LUV liposomes (20.4 ± 2.2%) was higher than that observed for MLV liposomes (7.5 ± 0.9%). Drug entrapment was 41.9 ± 6.2% and 27.2 ± 2.4% for PC and PC:CH LUV, respectively. The skin permeation was 1.55 ± 0.31%, 1.29 ± 0.40%, 0.20 ± 0.08%, and 0.50 ± 0.19% for PC LUV, PC:CH LUV, empty LUV + PA and aqueous solution, respectively. Controlled topical delivery, across stripped skin, was observed for PA entrapped in LUV liposomes.

Effect of gramicidin addition upon the physicochemical properties of dipalmitoyl phosphatidyl choline large unilamellar vesicles

In this work, we report the results bearing on the effect of gramicidin incorporation upon the dynamic and structural properties of dipalmitoyl phosphatidyl choline (DPPC) large unilamellar vesicle (LUVs). Results were obtained at different depths in the bilayer, both in the bulk of the bilayer and the vicinity of the added pentadecapeptide. This analysis was performed employing a series of extrinsic fluorescent probes (1,6-diphenyl-1,3,5-hexatriene (DPH), 1-(4-trimethylammonium phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), 6-dodecanoyl-2-dimethyl aminenaphthalene (Laurdan), pyrenedodecanoic acid, pyrene butyric acid, 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (Py–C 10–PC) and 1,2-bis-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (bis-Py–C 10–PC)). Fluorescence measurements were carried out after direct excitation of the probe or after its excitation by resonant energy transfer (RET) from the intrinsic tryptophan groups of the polypeptide. The last procedure allows an evaluation of the lipidic annulus properties in the vicinity of the incorporated polypeptide. In the gel state, gramicidin incorporation increases the order near the vesicle interface, as sensed by TMA-DPH. This increase is observed, above the phase transition temperature, over all the bilayer. This effect is probably related to a decrease in the lateral diffusion of the lipids, as evidenced by the reduced formation of intermolecular excimers following Py–C 10–PC excitation. Gramicidin incorporation also increases the penetration and/or mobility of water molecules located near the bilayer interface. In the gel phase, these changes are more relevant in the central part of the bilayer. When the LUVs are in the liquid crystalline state, the effect is observed through all the bilayer. In this state, the polypeptide incorporation also increases the diffusion/concentration of oxygen, irrespective of the probe localization. Resonance energy transfer between Trp residues and Laurdan, Py–C 10–PC or bis-Py–C 10–PC dyes was employed to evaluate the properties of the lipidic annulus surrounding the polypeptide. In the gel phase, gramicidin incorporation produces an increase in the penetration and/or dynamics of water molecules, while in the liquid crystalline state it leads to a decrease of the acyl chain mobility in the deepest parts of the bilayer.

The HIV-1 gp41 N-terminal heptad repeat plays an essential role in membrane fusion.

For many different enveloped viruses the crystal structure of the fusion protein core has been established. A striking conservation in the tertiary and quaternary arrangement of these core structures is repeatedly revealed among members of diverse families. It has been proposed that the primary role of the core involves structural rearrangements which facilitate apposition between viral and target cell membranes. Forming the internal trimeric coiled coil of the core, the N-terminal heptad repeat (NHR) of HIV-1 gp41 was suggested to have additional roles, due to its ability to bind biological membranes. The NHR is adjacent to the N-terminal hydrophobic fusion peptide (FP), which alone can fuse biological membranes. To investigate the role of the NHR in membrane fusion, we synthesized and functionally characterized HIV-1 gp41 peptides corresponding to the FP and NHR alone, as well as continuous peptides made of both FP and NHR (wild type and mutant). We show here that a consecutive, 70-residue peptide consisting of both the FP and NHR (gp41/1-70) has dramatic fusogenic properties. The effect of including the complete NHR, as compared to shorter 23-, 33-, or 52-residue N-terminal peptides, is illustrated by a leap in lipid mixing of phosphatidylcholine (PC) large unilamellar vesicles (LUV) and clearly delineates the synergistic role of the NHR in the fusion event. Furthermore, a mutation in the NHR that renders the virus noninfectious is reflected by a significant reduction in in vitro lipid mixing induced by the mutant, gp41/1-70 (I62D). Additional spectroscopic studies, characterizing membrane binding and apposition induced by the peptides, help to clarify the role of the NHR in membrane fusion.

Cholesterol modulates interaction between an amphipathic class A peptide, Ac-18A-NH2, and phosphatidylcholine bilayers.

Cholesterol (Chol) in phosphatidylcholine large unilamellar vesicles (PC LUV) modulated interaction of the bilayers with a class A amphipathic peptide, Ac-18A-NH2: Chol increased the peptide binding capacity and reduced the affinity together with the peptide-induced leakage of calcein from LUV. Similar effects of Chol have been observed on the interaction of LUV with apoA-I [Saito, H., Miyako, Y., Handa, T., and Miyajima, K. (1997) J. Lipid Res. 38, 287-294]. Circular dichroism (CD) spectra of the peptide indicated a similar helical structure formation in LUV with and without Chol. The fluorescence spectral shift, quantum yield, anisotropy, and acrylamide-quenching of the peptide Trp indicated that in PC:Chol (3:2) LUV, Ac-18A-NH2 was located in a more polar membrane environment with increased motional freedom and greater accessibility to the aqueous medium. Fluorescence energy transfer from the Trp indole ring to acceptors situated at different depths in the bilayers revealed that the amphipathic peptide penetrated the hydrophobic interior of PC bilayers, while the peptide was located at the polar zwitterionic surface in PC:Chol LUV. The inclusion of Chol causes the headgroup separation of PC at the surface of LUV and increases the binding maximum of the wedge-shaped amphipathic peptide without disrupting the membrane structure. In addition, the rigidifying effect of Chol on PC acyl chains prevents the penetration of the peptide into the bilayer interior. These findings imply that Chol in membranes affects the binding and motional freedom of exchangeable plasma apolipoproteins containing class A amphipathic sequences, e.g., apoA-I and apoCs.

Inhibitory effect of a quercetin metabolite, quercetin 3-O-beta-D-glucuronide, on lipid peroxidation in liposomal membranes.

To study the antioxidant activity of quercetin 3-O-beta-D-glucuronide (Q3GA), which is one of the quercetin metabolites in the blood after intake of quercetin-rich food, the inhibitory effect of Q3GA on lipid peroxidation was estimated using phosphatidylcholine large unilamellar vesicles (PC LUV) as a biomembrane model. Iron ion, an aqueous peroxyl radical generator, a peroxynitrite generator, or lipoxygenase was used as the inducer of lipid peroxidation. In all cases, Q3GA inhibited lipid peroxidation significantly, although its inhibitory effect was lower than that of quercetin aglycon. The ultrafiltration of PC LUV containing Q3GA revealed that Q3GA has low but significant affinity with the membranes of phospholipid bilayers. It is therefore likely that Q3GA acts as an efficient antioxidant in membranous lipid peroxidation through its localization in the phospholipid bilayer. This conjugated quercetin metabolite seems to retain the ability to protect cellular and subcellular membranes from peroxidative attack by reactive oxygen species and peroxidative enzymes.

A unique antioxidant activity of phosphatidylserine on iron-induced lipid peroxidation of phospholipid bilayers.

The relationship between the antioxidant effect of acidic phospholipids, phosphatidic acid (PA), phosphatidylglycerol (PG) and phosphatidylserine (PS), on iron-induced lipid peroxidation of phospholipid bilayers and their abilities to bind iron ion was examined in egg yolk phosphatidylcholine large unilamellar vesicles (EYPC LUV). The effect of each acidic phospholipid added to the vesicles at 10 mol% was assessed by measuring phosphatidylcholine hydroperoxides (PC-OOH) and thiobarbituric acid-reactive substances. The addition of dipalmitoyl PS (DPPS) showed a significant inhibitory effect, although the other two acidic phospholipids, dipalmitoyl PA (DPPA) and dipalmitoyl PG (DPPG), did not exert the inhibition. Neither dipalmitoyl PC (DPPC) nor dipalmitoyl phophatidylethanolamine (DPPE) showed any remarkable inhibition on this system. None of the tested phospholipids affected the lipid peroxidation rate remarkably when the vesicles were exposed to a water-soluble radical generator. The iron-binding ability of each phospholipid was estimated on the basis of the amounts of iron recovered in the chloroform/methanol phase after separation of the vesicle solution to water/methanol and chloroform/methanol phases. EYPC LUV containing DPPS, DPPA, and DPPG had higher amounts of bound iron than those containing DPPC and DPPE, indicating that these three acidic phospholipids possess an iron-binding ability at a similar level. Nevertheless, only DPPS suppressed iron-dependent decomposition of PC-OOH significantly. Therefore, it is likely that these three acidic phospholipids possess a significant iron-binding ability, although this ability per se does not warrant them antioxidative activities. The ability to suppress the iron-dependent decomposition of PC-OOH may explain the unique antioxidant activity of PS.

Resonance energy transfer study of peptide–lipid complexes

Resonance energy transfer involving tryptophan as a donor and anthrylvinyl-labeled phosphatidylcholine (AV-PC), 3-methoxybenzanthrone (MBA) and 8-anilino-1-naphthalene sulfonic acid (ANS) as acceptors has been examined to obtain information on the structure of peptide–lipid systems consisting of 18A or Ac-18A-NH 2 peptides and large unilamellar phosphatidylcholine vesicles. The lower and upper limits for the tryptophan distance from the bilayer midplane have been assessed in terms of the models of energy transfer in two-dimensional systems, taking into account orientational effects. Evidence for the existence of preferential orientations of Ac-18A-NH 2 with respect to the lipid–water interface has been obtained.

Interactions of Phosphatidylcholine Surface Monolayers with Triglyceride Cores and Enhanced ApoA-1 Binding in Lipid Emulsions

The binding maximum of apoA-1 (N) in triolein (TO)−egg yolk phosphatidylcholine (PC) emulsions was 10-fold larger than that in PC large unilamellar vesicles (LUV) of similar size (100 nm) with no significant difference in the affinity. Replacement of the long-chain triglyceride, TO, by medium-chain triglycerides or cholesteryl oleate in emulsion cores significantly decreased the N value. The 13C NMR chemical shifts of the PC carbonyl carbon at the surface layers indicated that PC polar headgroups are more separated and exposed to water molecules in emulsions than in vesicles. The N values were satisfactorily correlated with the chemical shift, that is, the degree of separation between the carbonyl groups at the surface. Although apoA-1 binding to the PC monolayers of emulsions brings about bending of the surface layers and creates local defects in the hydrocarbon regions in a similar manner as PC LUV, the surface−core interaction seems to fill the defects with the core neutral lipids, compensates for the ...

Plant fructans stabilize phosphatidylcholine liposomes during freeze-drying.

Fructans have been implicated as protective agents in the drought and freezing tolerance of many plant species. A direct proof of their ability to stabilize biological structures under stress conditions, however, is still lacking. Here we show that inulins (linear fructose polymers) isolated from chicory roots and dahlia tubers stabilize egg phosphatidylcholine large unilamellar vesicles during freeze-drying, while another polysaccharide, hydroxyethyl starch, was completely ineffective. Liposome stability was assessed after rehydration by measuring retention of the soluble fluorescent dye carboxyfluorescein and bilayer fusion. Inulin was an especially effective stabilizer in combination with glucose. Analysis by HPLC showed that the commercial inulin preparations used in our study contained no low molecular mass sugars that could be responsible for the observed stabilizing effect of the fructans. Fourier transform infrared spectroscopy showed a reduction of the gel to liquid-crystalline phase transition temperature of dry egg PtdCho by more than 20 degrees C in the presence of inulin. A direct interaction of inulin with the phospholipid in the dry state was also indicated by dramatic differences in the phosphate asymmetric stretch region of the infrared spectrum between samples with and without the polysaccharide.