Structure of Lipid Tubules Formed from a Polymerizable Lecithin

We have studied tubules formed from a polymerizable lipid in aqueous dispersion using freeze-fracture replication and transmission electron microscopy. The polymerizable diacetylenic lecithin 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine converts from liposomes to hollow cylinders, which we call tubules, on cooling through its chain melting phase transition temperature. These tubules differ substantially from cochleate cylinders formed by phosphatidylserines on binding of calcium. The tubules have diameters that range from 0.3 to 1 μm and lengths of up to hundreds of micrometers depending on conditions of formation. The thickness of the walls varies from as few as two bilayers to tens of bilayers in some longer tubules. Their surfaces may be either smooth, gently rippled, or with spiral steps depending on sample preparation conditions, including whether the lipids have been polymerized. The spiral steps may reflect the growth of the tubules by rolling up of flattened liposomes.     

Formation of age pigment-like fluorescent substances during peroxidation of lipids in model membranes

The formation of age pigment-like fluorescent substances during the lipid peroxidation of model membranes has been studied. Ferrous ion and ascorbate-induced lipid peroxidation of liposomal membranes containing phosphatidylethanolamine led to the formation of fluorescent substances which have characteristics similar to those of compounds derived from the reaction of phosphatidylethanolamine with purified fatty acid hydroperoxides. The fluorescent substances were accumulated in liposomal membranes, whereas thiobarbituric acid-reactive substances formed during lipid preoxidation were immediately released from the liposomal membranes. The thiobarbituric acid-reactive substances free from the membranes were not reactive with amino compounds such as phosphatidylethanolamine in liposomes or glycine in aqueous phase. It was suggested that the products reacting with amino compounds are short-lived, and may be rapidly inactivated after released into aqueous phase. The formation of fluorescent products was inefficient when phosphatidylethanolamine incorporated into the liposomes insensitive to lipid preoxidation was incubated with ferrous ion and ascorbate in the presence of liposomes sensitive to the peroxidation. The results suggest that some products generated from peroxidation-sensitive lipids react with the amino group of phosphatidylethanolamine molecules which are located on the same membranes, forming fluorescent substances. The presence of phosphatidylethanolamine in the membrane suppressed the formation of thiobarbituric acid-reactive substances, suggesting that phosphatidylethanolamine may react with radicals formed and terminate the propagation.     

Large-scale preparation of asymmetrically labeled fluorescent lipid vesicles

A method for producing lipid vesicles containing fluorescent phospholipid analogues localized to the inner leaflet of their membrane was developed. Incubation of a 450-fold molar excess of serum albumin with lipid vesicles symmetrically labeled with 1 mol % 1-palmitoyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazolyl)amino-caproyl phosphatidylcholine resulted in the removal of 99% of the fluorescent lipid from the outer leaflet. Asymmetrically labeled vesicles were separated from albumin/lipid complexes by gel filtration chromatography. Vesicles prepared in this manner were unable to transfer fluorescent lipid to cells during liposome-cell incubations. Liposomes asymmetrically labeled with other 4-nitrobenzo-2-oxa-1,3-diazole (NBD)-phospholipid analogues were also prepared. Removal of amino-dodecanoyl-NBD-labeled lipids from the outer leaflet of liposomes required three times more bovine serum albumin, and 48 h of incubation. This method can be used to produce large amounts of asymmetrically labeled liposomes suitable for use in investigating a variety of membrane phenomena.     

Enhancement of cell internalization and photostability of red and green emitter quantum dots upon entrapment in novel cationic nanoliposomes

Two quantum dots (QDs), a green emitter, CdSe and a red emitter, CdSe with ZnS shell are encapsulated into novel liposomes in two different formulations including cationic liposomes. Quantum dots have proven themselves as powerful inorganic fluorescent probes, especially for long‐term, multiplexed imaging and detection. Upon delivery into a cell, in endocytic vesicles such as endosomes, their fluorescence is quenched. We have investigated the potential toxic effects, photophysical properties and cell internalization of QDs in new formulation of liposomes as an in vitro vesicle model. Entrapment of QDs into liposomes is brought about with a decrease in their intrinsic fluorescence and toxicities and an increase in their photostability and lifetime. The biomimetic lipid bilayer of liposomes provides high biocompatibility, thereby enhancing the effectiveness of fluorescent nanoparticles for biological recognition in vitro and in vivo. The prepared lipodots could effectively prevent QDs from photo‐oxidation during storage and when exposed to ultraviolet (UV) light. Moreover, the flow cytometry of HEK 293 T cells showed that the cell internalization of encapsulated QDs in (DSPC/CHO/DOPE/DOAB) liposome is enhanced 10 times compared with non‐encapsulated QD (bare QDs).     

Quantitative Analysis of the Lamellarity of Giant Liposomes Prepared by the Inverted Emulsion Method

The inverted emulsion method is used to prepare giant liposomes by pushing water-in-oil droplets through the oil/water interface into an aqueous medium. Due to the high encapsulation efficiency of proteins under physiological conditions and the simplicity of the protocol, it has been widely used to prepare various cell models. However, the lamellarity of liposomes prepared by this method has not been evaluated quantitatively. Here, we prepared liposomes that were partially stained with a fluorescent dye, and analyzed their fluorescence intensity under an epifluorescence microscope. The fluorescence intensities of the membranes of individual liposomes were plotted against their diameter. The plots showed discrete distributions, which were classified into several groups. The group with the lowest fluorescence intensity was determined to be unilamellar by monitoring the exchangeability of the inner and the outer solutions of the liposomes in the presence of the pore-forming toxin α-hemolysin. Increasing the lipid concentration dissolved in oil increased the number of liposomes ∼100 times. However, almost all the liposomes were unilamellar even at saturating lipid concentrations. We also investigated the effects of lipid composition and liposome content, such as highly concentrated actin filaments and Xenopus egg extracts, on the lamellarity of the liposomes. Remarkably, over 90% of the liposomes were unilamellar under all conditions examined. We conclude that the inverted emulsion method can be used to efficiently prepare giant unilamellar liposomes and is useful for designing cell models.     

Embedding calix[4]resorcinarenes in liposomes: Experimental and computational investigation of the effect of resorcinarene inclusion on liposome properties and stability

Two calix[4]resorcinarenes, which differ in the length of the alkyl chain on the methylene bridge between the aromatic rings, have been embedded in unilamellar liposomes prepared from 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine in three host/guest ratios, following two different procedures. The effect of the insertion of the guests has been evaluated through the measurements of the viscosity and the kinetic stability of the liposomal systems by means of the fluorescent probes pyrene and 5(6)-carboxyfluorescein. The presence of the guests reduces the viscosity of the liposomes, suggesting a modification of the bilayer structure. However, this does not affect liposome stability. A calix[4]resorcinarene cavitand with a more rigid conformation compared to the parent resorcinarene, has been also synthetized and embedded in liposomes. The free energy of the insertion of the substrates in the lipid bilayer has been evaluated through Molecular Dynamics simulations.     

Quantitative Analysis of the Lamellarity of Giant Liposomes Prepared by the Inverted Emulsion Method

The inverted emulsion method is used to prepare giant liposomes by pushing water-in-oil droplets through the oil/water interface into an aqueous medium. Due to the high encapsulation efficiency of proteins under physiological conditions and the simplicity of the protocol, it has been widely used to prepare various cell models. However, the lamellarity of liposomes prepared by this method has not been evaluated quantitatively. Here, we prepared liposomes that were partially stained with a fluorescent dye, and analyzed their fluorescence intensity under an epifluorescence microscope. The fluorescence intensities of the membranes of individual liposomes were plotted against their diameter. The plots showed discrete distributions, which were classified into several groups. The group with the lowest fluorescence intensity was determined to be unilamellar by monitoring the exchangeability of the inner and the outer solutions of the liposomes in the presence of the pore-forming toxin α-hemolysin. Increasing the lipid concentration dissolved in oil increased the number of liposomes ∼100 times. However, almost all the liposomes were unilamellar even at saturating lipid concentrations. We also investigated the effects of lipid composition and liposome content, such as highly concentrated actin filaments and Xenopus egg extracts, on the lamellarity of the liposomes. Remarkably, over 90% of the liposomes were unilamellar under all conditions examined. We conclude that the inverted emulsion method can be used to efficiently prepare giant unilamellar liposomes and is useful for designing cell models.     

Permeability studies on liposomes formed from polymerizable diacetylenic phospholipids and their potential applications as drug delivery systems

We have investigated the permeability and entrapment characteristics of liposomes formed from a group of polymerizable phospholipids, containing diacetylenic groups in one or both of their acyl chains. Permeability was assessed by the release of an entrapped dye, 6-carboxyfluorescein. Diacetylenic phosphatidylcholine (PC) liposomes were found to exhibit a wide range of permeability properties, depending on: the nature of the diacetylenic lipid, i.e. mixed-chain (mc) or identical-chain (id), the extent of polymerisation, vesicle size, and cholesterol content. Ultraviolet-initiated polymerisation affected a significant decrease in the permeability of C25idPC liposomes. The increase in permeability of liposomes formed from four other diacetylenic lipids (C25mcPC, C23idPC, C23mcPC and C20idPC) after polymerisation was attributed to disturbances in the packing of lipid molecules, and/or the limited ability of small unilamellar vesicles to accommodate long polymers. The C20idPC lipid is atypical, forming irregular monomeric and polymeric vesicles. The permeability of C25idPC liposomes was also assessed by the release of [3H]inulin. C25idPC liposomes exhibited low permeabilities to [3H]inulin in their monomeric and polymeric states. Incubation of C25idPC liposomes in human plasma caused a substantial increase in the permeability of monomeric vesicles to both carboxyfluorescein and [3H]inulin. The permeability of polymerised C25idPC liposomes, however, was unaffected in the presence of plasma, with vesicles retaining most of their entrapped [3H]inulin after 50 h. These findings demonstrate that polymeric C25idPC liposomes exhibit high resistance to the destructive actions of plasma components, such as high-density lipoproteins (HDLs). Polymeric C25idPC liposomes may have an application in drug delivery systems.     

Modulation of bilayer structures derived from diacetylenic phosphocholines containing oxygen linker beta to diacetylene.

Phospholipids with diacetylenes present in the acyl chains form tubules and helices in aqueous dispersions. In order to modulate the morphology of bilayer structures and to understand the role of diacetylene in lipid-bilayer assembly, two diacetylenic phosphocholines, 1,2-bis(9,16-dioxa-hexacosa-11,13-diynoyl)-sn-3-phosph ocholine and 1,2-bis(15-oxa-pentacosa-10,12-diynoyl)-sn-3-phosphocholine, in which the diacetylene is linked to the acyl chain by an oxygen spacer have been synthesized. Lipid dispersions were characterized by calorimetric, film balance and microscopic techniques. Placement of oxygen spacer influences the morphology of the bilayer assemblies formed in aqueous solution. When both ends of the diacetylene were linked to the acyl chain by oxygen atoms, liposomes (diameters ranging from 0.3-3.4 microns) were observed by optical microscopy. Linking only the terminal portion of the acyl chain to the diacetylene with an oxygen atom resulted in a lipid which formed tubular microstructures as well as vesicles. Diameter of the tubular structures ranged from 0.4-4.7 microns. Transmission electron microscopic (TEM) analysis of replicas of a freeze fractured sample of the dispersion revealed that the tubular structures were hollow cylinders consisting of an aqueous core surrounded by a wall of lipid.     

Tethered fibronectin liposomes on supported lipid bilayers as a prepackaged controlled-release platform for cell-based assays

A biomimetic construct containing an extracellular matrix protein-liposome composite tethered on supported lipid bilayers (SLBs) was formed with fibronectin (FN), and polyethylene glycol (PEG) and cholesterol-containing liposomes. The construct can serve as a multifunctional platform for cell attachment and drug release. The successful fabrication of the FN-liposome-SLB model platform was analyzed in situ with a quartz crystal microbalance with dissipation. The long-term stability of the surface tethered liposomes was measured via an encapsulated fluorescent probe. Less than 20% of the fluorescent probe content was released in 8 days, which compared favorably to the release of 90% of the probe content in one day from a similar construct made without PEG and cholesterol. HeLa cells were used to study the cellular interactions with the model platform. The extracellular matrix composition, FN, was found to be essential to promote HeLa cell adhesion on the liposome-SLB surfaces. Upon cell adhesion, the liposomes were spatially reorganized and absorbed by the cells. The rate of HeLa cell apoptosis was correlated with the surface density of doxorubicin-loaded liposomes, confirming the effective drug delivery through liposomes. The multifunctional model platform could be useful as preadministered, controlled-release platforms for cell- and tissue-based assays.     

Comparison of fluorescence characteristics of products of peroxidation of membrane phospholipids with those of products derived from reaction of malonaldehyde with glycine as a model of lipofuscin fluorescent substances

The fluorescence characteristics of product (I), formed during the lipid peroxidation of rat liver phosphatidylcholine liposomes containing glycine, and fluorescent product (II), derived from the reaction of malonaldehyde with glycine, were examined to elucidate the mechanism of fluorescent chromophore formation. Fluorescent product (I) had a fluorescence emission maximum at 430 nm when excited at 360 nm; its fluorescence intensity decreases in alkaline medium, but is restored by readjustment of pH to neutrality. In contrast, fluorescent product (II) exhibited an emission maximum at 458 nm, and the fluorescence was quenched at acidic pH. The fluorescent substances formed during the lipid peroxidation of hemoglobin-free human erythrocyte ghost membranes had similar fluorescence characteristics to product (I). Gel filtration experiments showed that molecular size of fluorescent product (I) was larger than that of fluorescent product (II). The thiobarbituric acid-reactive substances released from peroxidizing liposomal phospholipids had a larger molecular size than malonaldehyde, and produced little or no fluorescence with glycine. It is concluded that the precursor of the fluorescent product formed during the lipid peroxidation of membrane phospholipids differs from malonaldehyde. The mechanism of the formation of blue emitting fluorescent material, believed to be a component of lipofuscin, seems to involve peroxidized phospholipids of the membrane.     

Influence des lecithines dicarboxyliques sur les liposomes

We have investigated the effect of dicarboxylic lecithins on liposomes prepared from egg-lecithins and their incorporation into liposome preparation.In the presence of phospholipids, liposome cell permeability to glucose is five times higher than in controls.The incorporation of dicarboxylic lecithins into liposomes at a molar concentration of 50 molecules per cent reduces by 60% the amount of glucose trapped inside the liposomes.Measurements of ovelecithin-glutaryl-lecithin liposomes stability show that stable structures are formed at a molar concentration of 70 molecules per cent in the presence of cholesterol. On the contrary, the liposomes prepared in the absence of cholesterol are not stable at a molar concentration of 50 molecules per cent.Measurements of sedimentation of mixed dispersions of monocarboxylic lecithins and glutaryl-lecithins show that glutaryl-lecithins must be in the form of micelles to solubilize the phosphatidyl cholines.     

Formation of helical protein assemblies of IgG and transducin on varied lipid tubules

Helical protein arrays on lipid tubules are valuable assemblies for studying protein structure and protein-lipid interactions through electron microscopy and crystallography. We describe conditions for forming such arrays from two proteins, IgG and transducin, the photoreceptor G protein, using a variety of lipid surfaces. Anti-dinitrophenyl (DNP) IgG arrays formed on DNP-phosphatidylethanolamine (DNP-PE) mixed with either galactosyl-ceramide lipids or phosphatidylcholine (PC) display different pH sensitivities and dimensions, yet have similar helical symmetries. DNP-PE/PC mixtures formed small crystals and large well-ordered flattened tubules. The peripheral membrane protein transducin (G(t)) formed helical arrays either on a mixture of cationic and neutral lipids or on residual photoreceptor lipids. Despite differences in lipid composition, the G(t) arrays have similar structures and show similar sensitivity to activation and variations in ionic environment. G(t) activation causes the helical assemblies to collapse to small vesicles, a process resembling the vesiculation of activated dynamin-lipid tubules. In a preliminary three-dimensional reconstruction, the hapten-bound IgG appears to make two contacts to the central lipid tubule, presumably via the F(ab) domains. The ability to generate a three-dimensional reconstruction without tilts illustrates one advantage of helical structures for two-dimensional crystallography, especially for visualizing protein-lipid interactions.     

Reaggregation of etioplast lipids and the formation of prolamellar bodies and thylakoids: An ultrastructural study

Etioplasts of dark-grownAvena sativa plants were used to prepare either saponin-free or saponin-containing prolamellar bodies. Lipid extracts from both fractions were studied in reaggregation experiments: extracts containing saponins showed liposomes as well as tubules, while saponin-free samples formed only liposomes. Purified PLB lipids in reaggregation experiments were either studied in the presence or in the absence of saponins. Best tubule formation was found with samples containing MGDG+saponin. However, the reconstruction of PLB-like structures was not possible. The long tubules, protruding from isolated PLBs, are seen as a result of the reaction of saponins (originally located in vacuoles) with MGDG. In memorian of Professor Shimon Klein     

Giant liposomes in physiological buffer using electroformation in a flow chamber

We describe a method to obtain giant liposomes (diameter 10-100 microm) in solutions of high ionic strength to perform a membrane-binding assay under physiological conditions. Using electroformation on ITO electrodes, we formed surface-attached giant liposomes in solutions of glycerol in a flow chamber and then introduced solutions of high ionic strength (up to 2 M KCl) into this chamber. The ionic solution exchanged with the isoosmolar glycerol solution inside and outside the liposomes. An initial mismatch in index of refraction between the inside and outside of liposomes allowed for the observation of solution replacement. Ions and small polar molecules exchanged into and out of surface-attached liposomes within minutes. In contrast, liposomes formed in solutions of macromolecules retained molecules larger than 4 kDa, allowing for encapsulation of these molecules for hours or days even if the solution outside the liposomes was exchanged. We propose that solutes entered liposomes through lipid tubules that attach liposomes to the film of lipids on the surface of the ITO electrode. The method presented here makes it straightforward to perform flow-through binding assays on giant liposomes under conditions of physiological ionic strength. We performed a membrane-binding assay for annexin V, a calcium-dependent protein that binds to phosphatidylserine (PS). The binding of annexin V depended on the concentration of PS and decreased as ionic strength increased to physiological levels.     

Giant liposomes in physiological buffer using electroformation in a flow chamber

We describe a method to obtain giant liposomes (diameter 10-100 μm) in solutions of high ionic strength to perform a membrane-binding assay under physiological conditions. Using electroformation on ITO electrodes, we formed surface-attached giant liposomes in solutions of glycerol in a flow chamber and then introduced solutions of high ionic strength (up to 2 M KCl) into this chamber. The ionic solution exchanged with the isoosmolar glycerol solution inside and outside the liposomes. An initial mismatch in index of refraction between the inside and outside of liposomes allowed for the observation of solution replacement. Ions and small polar molecules exchanged into and out of surface-attached liposomes within minutes. In contrast, liposomes formed in solutions of macromolecules retained molecules larger than 4 kDa, allowing for encapsulation of these molecules for hours or days even if the solution outside the liposomes was exchanged. We propose that solutes entered liposomes through lipid tubules that attach liposomes to the film of lipids on the surface of the ITO electrode. The method presented here makes it straightforward to perform flow-through binding assays on giant liposomes under conditions of physiological ionic strength. We performed a membrane-binding assay for annexin V, a calcium-dependent protein that binds to phosphatidylserine (PS). The binding of annexin V depended on the concentration of PS and decreased as ionic strength increased to physiological levels.     

Morphology of Cationic Liposome/DNA Complexes in Relation to Their Chemical Composition

Abstract

Electron microscopy is used to show the morphology of liposome/DNA complexes as related to their cationic component, the molar ratio of the helper lipid (usually DOPE¹), the nature of the DNA-component, as well as the composition of the media. Liposomes made of monovalent cationic amphiphiles adhere and fuse during interaction with negatively charged DNA thereby complexing the DNA. The size of the resulting complexes is depending upon charge neutralization and is smallest at a slightly positive net charge. At molar ratios of DOPE, to the cationic component of ≥ 1.5, hexagonal lipid tubules are formed, especially in media containing high salt concentrations, and even in the control lipid mixture, not interacting with any DNA or oligonucleotide. Complexes, made of plasmid-DNA, monovalent cationic amphiphiles, and DOPE at a lower molar ratio, show additionally to the semifused or fused liposomes a new structure, called spaghetti-like structure, representing a bilayer-coated, supercoiled DNA. Single-strand and short oligonucleotides seem not to form such structures during interaction with monovalent cationic liposomes. Neither fusion nor spaghetti formation is observed during interaction of DNA with liposomes made of polyvalent cationic amphiphiles. In general, small complexes consisting of some few semifused liposomes bearing the self-encapsulated nucleic acid and additionally the spaghetti-like structure, free or connected with these complexes, seem to be candidates for the transfectionactive structure rather than large extended H_II¹-lipid arrangements.     

Interaction analysis of a ladder-shaped polycyclic ether and model transmembrane peptides in lipid bilayers by using Förster resonance energy transfer and polarized attenuated total reflection infrared spectroscopy

Ladder-shaped polycyclic ethers (LSPs) are predicted to interact with membrane proteins; however, the underlying mechanism has not been satisfactorily elucidated. It has been hypothesized that LSPs possess non-specific affinity to α-helical segments of transmembrane proteins. To verify this hypothesis, we constructed a model LSP interaction system in a lipid bilayer. We prepared 5 types of α-helical peptides and reconstituted them in liposomes. The reconstitution and orientation of these peptides in the liposomes were examined using polarized attenuated total reflection infrared (ATR-IR) spectroscopy and gel filtration. The results revealed that 4 peptides were retained in liposomes, and 3 of them formed stable transmembrane structures. The interaction between the LSP and the peptides was investigated using Förster resonance energy transfer (FRET). In the lipid bilayer, the LSP strongly recognized the peptides that possessed aligned hydrogen donating groups with leucine caps. We propose that this leucine-capped 16-amino acid sequence is a potential LPS binding motif.     

Physical properties of liposomes and proteoliposomes prepared from Escherichia coli polar lipids

Reconstituted proteoliposomes serve as experimental systems for the study of membrane enzymes. Osmotic shifts and other changes in the solution environment may influence the structures and membrane properties of phospholipid vesicles (including liposomes, proteoliposomes and biological membrane vesicles) and hence the activities of membrane-associated proteins. Polar lipid extracts from Escherichia coli are commonly used in membrane protein reconstitution. The solution environment influenced the phase transition temperature and the diameter of liposomes and proteoliposomes prepared from E. coli polar lipid by extrusion. Liposomes prepared from E. coli polar lipids differed from dioleoylphosphatidylglycerol liposomes in Young's elastic modulus, yield point for solute leakage and structural response to osmotic shifts, the latter indicated by static light scattering spectroscopy. At high concentrations, NaCl caused aggregation of E. coli lipid liposomes that precluded detailed interpretation of light scattering data. Proteoliposomes and liposomes prepared from E. coli polar lipids were similar in size, yield point for solute leakage and structural response to osmotic shifts imposed with sucrose as osmolyte. These results will facilitate studies of bacterial enzymes implicated in osmosensing and of other enzymes that are reconstituted in E. coli lipid vesicles.