1-2-oleoylphosphatidylcholine and fluorexon

Novel polymers containing quaternary functional groups, with and without (control copolymer) PEG side chains, were synthesized and characterized for their ability to lyse the phospholipid membranes of liposome vesicles. Calcein loaded unilamellar vesicles composed of 1,2-dioleoyl- sn-glycero-3-phosphatidylcholine (DOPC) were used to mimic red-blood cell membranes, and a 80:20 (mol/mol) mixture of 1,2-dioleoyl- sn-glycero-3-phosphatidyl ethanolamine (DOPE) and 1,2-dioleoyl- sn- glycero-3-[phospho- rac-(1-glycerol)] (DOPG) was used to mimic the outer cell-membrane of the gram-negative bacteria, E. coli. For DOPE/DOPG = 80:20 (mol/mol) liposome vesicles, the PEG bottle brush copolymer caused leakage of the encapsulated Calcein dye, whereas the control copolymer did not cause any leakage. Both the bottle brush copolymer and the copolymer without PEG side chains had no effect on the zwitterionic DOPC liposome vesicles indicating that the RBC membrane composition is not disrupted by either copolymer architecture. The PEG bottle brush copolymer did not affect the colloidal size of the DOPE/DOPG = 80:20 (mol/mol) liposome vesicles, but on the addition of Triton-X 100, the vesicles disappeared. This provided evidence that the dye leakage was caused by compromising the integrity of the vesicle membrane by the bottle brush polymer architecture. Such partial disruption was preceded by the entropic templating of lipid membranes by the PEG side chains of the bottle brush copolymer. By careful comparison with non-PEGylated cationic polymers, Quart, the importance of PEG side chains in the membrane disrupting activity of the PEGylated cationic polymer, QPEG, was demonstrated. This finding itself is interesting and can contribute to the expansion of the design of membrane disrupting materials.

Topics: Coloring Agents; Fluoresceins; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Polyamines; Polyethylene Glycols; Unilamellar Liposomes

Highly parallel measurements on single, tethered lipid vesicles enable real-time monitoring of dynamic membrane interactions of relevance to medical, pharmaceutical, and biotechnological applications. Monitoring the time-dependent release of entrapped fluorescent dyes is a popular measurement approach, although it is often challenging to accurately extract quantitative biochemical parameters. Key issues include dye leakage and imaging-related photobleaching, and corrective measures are needed. Herein, we present an extended analytical framework to collect and interpret time-lapsed fluorescence microscopy imaging data, and demonstrate its utility for tracking membrane-peptide interactions. Our approach is focused on improving platform design and data analysis. First, we identified suitable membrane compositions to minimize dye leakage while enhancing the biomimetic character of lipid vesicles. Second, a data normalization procedure was developed to correct for experimental artifacts, namely dye leakage and photobleaching, and hence improve measurement accuracy. This analytical procedure was applied to experimentally determine the rate of peptide-induced pore formation in single lipid vesicles, and there was up to a nearly three-fold decrease in the measured rate, as compared to uncorrected data. Taken together, the results present a broadly applicable analytical framework to account for experimental artifacts and improve measurement accuracy in highly parallel, single lipid vesicle arrays.

Topics: Amino Acid Sequence; Artifacts; Cholesterol; Diffusion; Fluoresceins; Fluorescent Dyes; Kinetics; Lipid Bilayers; Microscopy, Fluorescence; Peptides; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Photobleaching; Polyethylene Glycols; Rhodamines; Time-Lapse Imaging; Unilamellar Liposomes

Non-fluidic bio-layer interferometry (BLI) has rapidly become a standard tool for monitoring almost all biomolecular interactions in a label-free, real-time and high-throughput manner. High-efficiency screening methods which measure the kinetics of liposomes with a variety of compounds require the immobilization of liposomes. In this work, a method is described for immobilizing liposomes for interaction studies, based on the biophysical principles of this biosensor platform. The immobilization approach includes the loading of DSPE-PEG

Topics: 1,2-Dipalmitoylphosphatidylcholine; Adsorption; Biosensing Techniques; Biotin; Cardiolipins; Cytochromes c; Drosophila Proteins; Fluoresceins; Fluorescent Dyes; High-Throughput Screening Assays; Hydrophobic and Hydrophilic Interactions; Interferometry; Kinetics; Liposomes; Micelles; Microscopy, Fluorescence; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Polyethylene Glycols; Protein Phosphatase 1; Reproducibility of Results

Photo-triggerable liposomes are considered nowadays as promising drug delivery devices due to their potential to release encapsulated drugs in a spatial and temporal manner. In this work, we have investigated the photopermeation efficiency of three photosensitizers (PSs), namely verteporfin, pheophorbide a and m-THPP when incorporated into liposomes with well-defined lipid compositions (SOPC, DOPC or SLPC). By changing the nature of phospholipids and PSs, the illumination of the studied systems was shown to significantly alter their lipid bilayer properties via the formation of lipid peroxides. The system efficiency depends on the PS/phospholipid association, and the ability of the PS to peroxidize acyl chains. Our results demonstrated the possible use of these three clinically approved (or under investigation) PSs as potential candidates for photo-triggerable liposome conception.

Topics: Chlorophyll; Drug Liberation; Fluoresceins; Fluorescent Dyes; Hydrophobic and Hydrophilic Interactions; Light; Lipid Bilayers; Lipid Peroxidation; Liposomes; Mesoporphyrins; Molecular Dynamics Simulation; Permeability; Phosphatidylcholines; Photosensitizing Agents; Porphyrins; Transition Temperature; Verteporfin

OSW-1 is a structurally unique steroidal saponin isolated from the bulbs of Ornithogalum saundersiae, and has exhibited highly potent and selective cytotoxicity in tumor cell lines. This study aimed to investigate the molecular mechanism for the membrane-permeabilizing activity of OSW-1 in comparison with those of other saponins by using various spectroscopic approaches. The membrane effects and hemolytic activity of OSW-1 were markedly enhanced in the presence of membrane cholesterol. Binding affinity measurements using fluorescent cholestatrienol and solid-state NMR spectroscopy of a 3-d-cholesterol probe suggested that OSW-1 interacts with membrane cholesterol without forming large aggregates while 3-O-glycosyl saponin, digitonin, forms cholesterol-containing aggregates. The results suggest that OSW-1/cholesterol interaction is likely to cause membrane permeabilization and pore formation without destroying the whole membrane integrity, which could partly be responsible for its highly potent cell toxicity.

Topics: Antineoplastic Agents, Phytogenic; beta-Cyclodextrins; Biological Transport; Cholestenones; Cholesterol; Digitonin; Dimyristoylphosphatidylcholine; Erythrocyte Membrane; Fluoresceins; Glycyrrhizic Acid; Hemolysis; Humans; Membrane Lipids; Oleanolic Acid; Ornithogalum; Phosphatidylcholines; Saponins; Unilamellar Liposomes

Enzymatic digestion of bovine lactoferrin generates lactoferricin B (Lfcin B), a 25-mer peptide with strong antimicrobial activity of unknown mechanism. To elucidate the mechanistic basis of Lfcin B bactericidal activity, we investigated the interaction of Lfcin B with Escherichia coli and liposomes of lipid membranes. Lfcin B induced the influx of a membrane-impermeant fluorescent probe, SYTOX green, from the outside of E. coli into its cytoplasm. Lfcin B induced gradual leakage of calcein from large unilamellar vesicles (LUVs) of dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) membranes. To clarify the cause of Lfcin B-induced leakage of calcein from the LUVs, we used the single giant unilamellar vesicle (GUV) method to investigate the interaction of Lfcin B with calcein-containing DOPG/DOPC-GUVs. We observed that a rapid leakage of calcein from a GUV started stochastically; statistical analysis provided a rate constant for Lfcin B-induced pore formation, kp. On the other hand, phase-contrast microscopic images revealed that Lfcin B induced a rapid leakage of sucrose from the single GUVs with concomitant appearance of a spherical GUV of smaller diameter. Because of the very fast leakage, and at the present time resolution of the experiments (33 ms), we could not follow the evolution of pore nor the process of the structural changes of the GUV. Here we used the term "local rupture" to express the rapid leakage of sucrose and determined the rate constant of local rupture, kL. On the basis of the comparison between kp and kL, we concluded that the leakage of calcein from single GUVs occurred as a result of a local rupture in the GUVs and that smaller pores inducing leakage of calcein were not formed before the local rupture. The results of the effect of the surface charge density of lipid membranes and that of salt concentration in buffer on kp clearly show that kp increases with an increase in the extent of electrostatic interactions due to the surface charges. Analysis of Lfcin B-induced shape changes indicated that the binding of Lfcin B increased the area of the outer monolayer of GUVs. These results indicate that Lfcin B-induced damage of the plasma membrane of E. coli with its concomitant rapid leakage of internal contents is a key factor for the bactericidal activity of LfcinB.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Cattle; Cell Membrane Permeability; Escherichia coli; Escherichia coli Infections; Fluoresceins; Fluorescent Dyes; Humans; Lactoferrin; Molecular Sequence Data; Organic Chemicals; Phosphatidylcholines; Phosphatidylglycerols; Static Electricity; Sucrose; Unilamellar Liposomes

Surfactin, a bacterial amphiphilic lipopeptide is attracting more and more attention in view of its bioactive properties which are in relation with its ability to interact with lipids of biological membranes. In this work, we investigated the effect of surfactin on membrane structure using model of membranes, vesicles as well as supported bilayers, presenting coexistence of fluid-disordered (DOPC) and gel (DPPC) phases. A range of complementary methods was used including AFM, ellipsometry, dynamic light scattering, fluorescence measurements of Laurdan, DPH, calcein release, and octadecylrhodamine B dequenching. Our findings demonstrated that surfactin concentration is critical for its effect on the membrane. The results suggest that the presence of rigid domains can play an essential role in the first step of surfactin insertion and that surfactin interacts both with the membrane polar heads and the acyl chain region. A mechanism for the surfactin lipid membrane interaction, consisting of three sequential structural and morphological changes, is proposed. At concentrations below the CMC, surfactin inserted at the boundary between gel and fluid lipid domains, inhibited phase separation and stiffened the bilayer without global morphological change of liposomes. At concentrations close to CMC, surfactin solubilized the fluid phospholipid phase and increased order in the remainder of the lipid bilayer. At higher surfactin concentrations, both the fluid and the rigid bilayer structures were dissolved into mixed micelles and other structures presenting a wide size distribution.

Topics: 1,2-Dipalmitoylphosphatidylcholine; 2-Naphthylamine; Bacterial Physiological Phenomena; Calorimetry; Cell Membrane; Diphenylhexatriene; Fluoresceins; Laurates; Light; Lipid Bilayers; Lipids; Lipopeptides; Liposomes; Micelles; Microscopy, Atomic Force; Microscopy, Fluorescence; Peptides, Cyclic; Phosphatidylcholines; Phospholipids; Rhodamines; Scattering, Radiation; Spectrometry, Fluorescence

Phospholipase C (PLC) is considered to be one of key enzymes for the design of drug delivery system using the endocytosis route, because PLC can catalyze the membrane fusion between cell membranes and phospholipid vehicles (liposomes). Membrane fusion by PLC was then studied under various pHs to model the endosomal environment. The used liposomes were composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and cholesterol (Ch). The membrane fusion was dominated by the enzymatic reaction at pH 6-7.5. In contrast, the membrane perturbation effect due to the conformational change of PLC could induce the membrane fusion at around pH 4. The maximal value of membrane fusion was observed at around pH 5 for three liposomes in the order of DOPC

Topics: 1,2-Dipalmitoylphosphatidylcholine; Bacillus cereus; Cholesterol; Circular Dichroism; Endosomes; Fluoresceins; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Ligands; Liposomes; Membrane Fusion; Phosphatidylcholines; Protein Structure, Secondary; Protein Structure, Tertiary; Spectrometry, Fluorescence; Type C Phospholipases

The mechanism of pore formation of lytic peptides, such as melittin from bee venom, is thought to involve binding to the membrane surface, followed by insertion at threshold levels of bound peptide. We show that in membranes composed of zwitterionic lipids, i.e. phosphatidylcholine, melittin not only forms pores but also inhibits pore formation. We propose that these two modes of action are the result of two competing reactions: direct insertion into the membrane and binding parallel to the membrane surface. The direct insertion of melittin leads to pore formation, whereas the parallel conformation is inactive and prevents other melittin molecules from inserting, hence preventing pore formation.

Topics: Animals; Bee Venoms; Bees; Cell Membrane; Circular Dichroism; Dose-Response Relationship, Drug; Fluoresceins; Lipids; Liposomes; Melitten; Molecular Conformation; Phosphatidylcholines; Protein Structure, Tertiary; Surface Properties

Listeriolysin O (LLO) is the most important virulence factor of the intracellular pathogen Listeria monocytogenes. Its main task is to enable escape of bacteria from the phagosomal vacuole into the cytoplasm. LLO belongs to the cholesterol-dependent cytolysin (CDC) family but differs from other members, as it exhibits optimal activity at low pH. Its pore forming ability at higher pH values has been largely disregarded in Listeria pathogenesis. Here we show that high cholesterol concentrations in the membrane restore the low activity of LLO at high pH values. LLO binds to lipid membranes, at physiological or even slightly basic pH values, in a cholesterol-dependent fashion. Binding, insertion into lipid monolayers, and permeabilization of calcein-loaded liposomes are maximal above approximately 35 mol % cholesterol, a concentration range typically found in lipid rafts. The narrow transition region of cholesterol concentration separating low and high activity indicates that cholesterol not only allows the binding of LLO to membranes but also affects other steps in pore formation. We were able to detect some of these by surface plasmon resonance-based assays. In particular, we show that LLO recognition of cholesterol is determined by the most exposed 3beta-hydroxy group of cholesterol. In addition, LLO binds and permeabilizes J774 cells and human erythrocytes in a cholesterol-dependent fashion at physiological or slightly basic pH values. The results clearly show that LLO activity at physiological pH cannot be neglected and that its action at sites distal to cell entry may have important physiological consequences for Listeria pathogenesis.

Topics: Bacterial Toxins; Cell Membrane; Cell Membrane Permeability; Cholesterol; Dimerization; Dose-Response Relationship, Drug; Erythrocyte Membrane; Fluoresceins; Fluorescent Dyes; Heat-Shock Proteins; Hemagglutinins; Hemolysin Proteins; Humans; Hydrogen-Ion Concentration; Liposomes; Listeria monocytogenes; Membrane Microdomains; Molecular Structure; Phosphatidylcholines; Porosity; Protein Binding; Sterols; Surface Plasmon Resonance; Virulence Factors

Topics: Bacterial Proteins; Biosensing Techniques; Drug Delivery Systems; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Membrane Proteins; Phosphatidylcholines; Protons

Protein aggregation is central to most neurodegenerative diseases, as shown by familial case studies and by animal models. A modified 'amyloid cascade' hypothesis for Alzheimer's disease states that prefibrillar oligomers, also called amyloid-beta-derived diffusible ligands or globular oligomers, are the responsible toxic agent. It has been proposed that these oligomeric species, as shown for amyloid-beta, beta2-microglobulin or prion fragments, exert toxicity by forming pores in membranes, initiating a cascade of detrimental events for the cell. Interaction of granular aggregates and globular oligomers of an amyloidogenic protein, human stefin B, with model lipid membranes and monolayers was studied. Prefibrillar oligomers/aggregates of stefin B are shown to cause concentration-dependent membrane leaking, in contrast to the homologous stefin A. Prefibrillar oligomers/aggregates of stefin B also increase the surface pressure at an air-water interface, i.e. they have amphipathic character and are surface seeking. In addition, they show stronger interaction with 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] monolayers than native stefin A or nonaggregated stefin B. Prefibrillar aggregates interact predominantly with acidic phospholipids, such as dioleoylphosphatidylglycerol or dipalmitoylphosphatidylserine, as shown by calcein release experiments and surface plasmon resonance. The same preparations are toxic to neuroblastoma cells, as determined by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, again in contrast to the homologue stefin A, which does not aggregate under any of the conditions studied. This study is aimed to contribute to the general model of cellular toxicity induced by prefibrillar oligomers of amyloidogenic proteins, not necessarily involved in pathology.

Topics: Cell Membrane; Cystatin A; Cystatin B; Cystatins; Cysteine Proteinase Inhibitors; Fluoresceins; Humans; Lipid Bilayers; Membrane Lipids; Neuroblastoma; Neurodegenerative Diseases; Neurofibrillary Tangles; Permeability; Phosphatidylcholines; Phosphatidylglycerols; Surface Properties; Tetrazolium Salts; Thiazoles; Toxicity Tests; Tumor Cells, Cultured

Photochemical switching has been studied of double-tailed phosphate amphiphiles containing azobenzene units in both tails in aqueous vesicular dispersions and in mixed vesicular systems with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Since the ease of switching depends on the strength of the bilayer packing, particular emphasis has been placed on the occurrence of H-aggregation in the hydrophobic core of the vesicles. UV-vis spectrometry was employed to monitor H-aggregation and showed how this process depends on the ionic strength and on the mode of preparation of the vesicles. Two types of H-aggregates were observed in mixed DOPC vesicles with 5 mol % of azobenzene phosphate: one with lambda(max) at around 300 nm and one with lambda(max) at 305-320 nm. Those with lambda(max) at 300 nm could not be trans-cis photoisomerized, whereas those with lambda(max) at 305-320 nm are more loosely packed and can be photochemically switched. The permeability of the vesicular bilayers, as probed with leakage experiments using calcein as a fluorescent probe, was examined as another measure for the strength of bilayer packing. Leakage occurred only for DOPC vesicles containing more than 20 mol % of azobenzenephosphate, irradiated with UV light to induce trans-cis photoisomerization. We contend that detailed information on bilayer packing will be of crucial importance for fine-tuning the lateral pressure in vesicular membranes with the ultimate aim to steer the opening and closing of mechanosensitive protein channels of large conductance.

Topics: Azo Compounds; Calorimetry, Differential Scanning; Escherichia coli Proteins; Fluoresceins; Hydrogen; Ion Channels; Light; Lipid Bilayers; Lipids; Liposomes; Membrane Fluidity; Membrane Lipids; Membranes, Artificial; Microscopy, Electron, Transmission; Models, Chemical; Phosphates; Phosphatidylcholines; Phospholipids; Photochemistry; Spectrophotometry, Ultraviolet; Temperature; Ultraviolet Rays

The pH dependence of the antimicrobial and membrane activity of clavanin A, a peptide antibiotic that is rich in histidines and glycines, was analyzed in growth and membrane leakage experiments. Clavanin A more effectively inhibited the growth of the test organism Lactobacillus sake when the pH of the medium was lowered. Whereas the wild-type peptide efficiently released fluorophores from unilamellar vesicles at neutral pH according to a nonspecific permeabilization mechanism, it did not permeabilize model bilayers at low pH. It was therefore suggested that this peptide uses a distinct mode of action under acidic conditions different than that used around neutral pH. However, at low pH, the membrane is still the target for clavanin A, as the peptide collapsed both vital transmembrane proton gradients and ion gradients under these conditions. Clavanin A did not act as a ionophore across phospholipid bilayers, indicating that membrane constituents other than membrane phospholipids are involved in the dissipation of transmembrane ion gradients. Membrane proteins that generate transmembrane ion gradients are suggested to be the targets for clavanin A at low pH. In addition to the histidines, the three glycine residues of clavanin A are shown to play an important role in the specific mode of interaction with these membrane targets. These residues may induce a flexible hydrophobic conformation that allows the peptide to exert different membrane activities. This study demonstrates that clavanin A is a special membrane-active peptide that has access to two markedly distinct pH-dependent modes of actions.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Biological Transport; Blood Proteins; Cell Membrane Permeability; Circular Dichroism; Fluoresceins; Hydrogen-Ion Concentration; Lactobacillus; Lipid Bilayers; Liposomes; Models, Molecular; Molecular Sequence Data; Phosphatidylcholines; Potassium; Protein Conformation; Protons

The effects of glucose and its oligomers (maltodextrins) on the stability of sonicated liposomes during freeze-drying were studied by monitoring the retention of the fluorescent dye, Calcein, entrapped in the liposomal inner aqueous phase and by the use of differential scanning calorimetry (DSC). Glucose showed weak cryoprotective effects on dioleoylphosphatidylcholine (DOPC) or egg yolk phosphatidylcholine (eggPC) liposomes, while it had a relatively high cryoprotective effect on dipalmitoylphosphatidylcholine (DPPC) liposomes. Maltose and maltotriose showed high cryoprotective effects on eggPC liposomes, while other maltodextrin, longer oligomers, showed low cryoprotective effects. No saccharide was effective to protect DOPC liposomes. The fluidity and/or packing of lipid membranes had considerable influences on the stability of liposomes during the lyophilization. Maltodextrins showed relatively high cryoprotective effects on DPPC liposomes at low saccharide/lipid molar ratios, although the cryoprotective effects decreased with the increase in the molar ratios. Size measurements suggested that glucose and maltose completely prevented the aggregation and/or fusion of liposomes during lyophilization, and that other maltodextrins induced them due to their weak hydrophobic properties.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Calorimetry, Differential Scanning; Cryoprotective Agents; Drug Stability; Fluoresceins; Fluorescent Dyes; Freeze Drying; Glucose; Liposomes; Membrane Fluidity; Phosphatidylcholines; Polysaccharides; Sonication

The stability of two-component liposomes composed of the polymerizable 1,2-bis-[10-(2',4'-hexadienoyloxy)decanoyl]-sn-glycero-3-phosphati dylcholine (SorbPC) and either a phosphatidylethanolamine (PE) or a phosphatidylcholine (PC) were examined via fluorescence leakage assays. Ultraviolet light exposure of SorbPC-containing liposomes forms poly-SorbPC, which phase separates from the remaining monomeric lipids. If the nonpolymerizable lipids are PE's, then the photoinduced polymerization destabilizes the liposome with loss of aqueous contents. The permeability of the control dioleoylPC/SorbPC membranes was not affected by photopolymerization of SorbPC. The photodestabilization of dioleoylPE/SorbPC (3:1) liposomes required the presence of oligolamellar liposomes. NMR spectroscopy of extended bilayers of dioleoylPE/SorbPC (3:1) showed that the photopolymerization lowers the temperature for the appearance of 31P NMR signals due to the formation of isotropically symmetric lipid structures. These observations suggest the following model for the photoinduced destabilization of liposomes composed of PE/SorbPC; photopolymerization induced phase separation with the formation of enriched domains of PE, which allows the close approach of apposed regions of enriched PE lamellae and permits the formation of an isotropically symmetric structure between the lamellae. The formation of such an interlamellar attachment (ILA) between the lamellae of an oligolamellar liposome provides a permeability pathway for the light-stimulated leakage of entrapped water-soluble reagents.

Topics: Drug Stability; Fluoresceins; Liposomes; Magnetic Resonance Spectroscopy; Models, Structural; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Photolysis; Structure-Activity Relationship; Ultraviolet Rays

The importance of the protein-linked carbohydrates for the stabilization of dioleoylphosphatidylethanolamine (DOPE) bilayers has been investigated using glycophorin A, the major sialoglycoprotein of the human erythrocyte membrane, as a stabilizer. Two major types of glycophorin, differing in the sialic acid content, were used in the study. Type MM contains 19.2 +/- 2.5 sialic residues per molecule of glycophorin, and type NN contains 10.8 +/- 1.2. Type MM could stabilize DOPE bilayers at 0.5 mol%, whereas type NN was unable to do so even at 1 mol%. The importance of the sialic acid content to the stabilization activity of glycophorin was further confirmed by the observation that the neuraminidase-treated type MM showed a lower stabilization activity than the untreated type. Since type NN had no stabilizing activity, we attempted to couple a trisaccharide, NeuNAc----Gal----Glc, to type NN by reductive amination. 2.5 +/- 0.8 saccharide chains were added per molecule of type NN. The trisaccharide-attached type NN showed a greater stabilization activity than the parent type NN molecule, indicating again that the sialic acid content of the stabilizer molecule determines the stabilization activity. Addition of wheat-germ agglutinin (WGA), which binds to the sialic acid residues of a glycoprotein, to type MM-stabilized liposomes caused rapid aggregation and destabilization of liposomes, resulting in leakage of an entrapped marker, calcein. The aggregation increased with increasing amount of the lectin; however, the leakage rate was maximum at an optimum concentration of WGA. These results are discussed in terms of the role of sialic acid in the interfacial hydration and charge repulsion which determines the DOPE bilayer stability.

Topics: Carbohydrate Sequence; Chromatography, Affinity; Fluoresceins; Glycophorins; Humans; Indicators and Reagents; Lipid Bilayers; Molecular Sequence Data; Molecular Weight; Oligosaccharides; Phosphatidylcholines; Phosphatidylethanolamines; Protein Binding; Sialoglycoproteins