Cationic polymer adsorption on bilayer membrane containing anionic and cationic lipids

Microelectrophoresis, dynamic light scattering, fluorescence, and microcalorimetry are used to study the adsorption of a synthetic polycation, poly-N-ethyl-4-vinylpyridinium bromide, on the surface of three-component liposomes formed from electrically neutral phosphatidylcholine, anionic diphosphatidylglycerol (cardiolipin), and cationic dicetyldimethylammonium bromide, with the two latter being taken in equal amounts. The adsorption of the polycation on the liposomal membrane results in the generation of a positive charge, which provides the polycation-liposome complex with aggregation stability. Increasing salt concentration in the suspension causes the complex to dissociate into its components. According to the microcalorimetry data, the membranes of the initial three-component liposomes consist of two microphases, with one of them being enriched with the neutral lipid and another one, with the ionic components. The polycation adsorption does not lead to noticeable structural rearrangements in the liposomal membranes.     

Structure and properties of complexes formed by cationic polymers and anionic cholesterol-containing liposomes

The adsorption of the synthetic polycation poly(N-ethyl-4-vinylpyridinium bromide) on the surface of three-component lipid vesicles (liposomes) formed from a mixture of anionic cardiolipin, electroneutral egg lecithin, and nonionic cholesterol is studied via laser microelectropheresis, dynamic light scattering, conductometry, fluorescence spectroscopy, and UV spectroscopy. The incorporation of cholesterol into the liposomal membrane increases its microviscosity; however, the membrane remains liquid-crystalline. Simultaneously, an increase in the fraction of cholesterol causes the formation of defects in liposome membranes during their binding with poly(N-ethyl-4-vinylpyridium bromide) and makes complexation irreversible. The results of this study are of interest for predicting the behavior of polyelectrolytes and biologically active structures formed on their basis on the surface of cells and the reaction of the cellular membrane to the adsorbed polymer.     

Complexes of a cationic polymer with oxidized anionic liposomes: Composition,structure, and properties

Formation of complexes obtained by the adsorption of a cationic polymer, poly(N-ethyl-4-vinylpyridium bromide), with a degree of polymerization of 600 on the surface of 50-nm bilayer vesicles (liposomes) formed from neutral phosphatidyl choline, anionic diphosphatidyl glycerol (cardiolipin), and a surfactant with one alkyl radical, such as electroneutral n-hexadecylphosphocholine, palmitic acid, or heptanoic acid, is studied. The incorporation of these surfactants into the liposomal membrane stimulates the appearance of oxidized forms of lipids in it. The incorporation of n-hexadecylphosphocholine into the membrane of n-hexadecylphosphocholine and palmitic acid with the alkyl radical, whose length is comparable with the length of alkyl radicals in a lipid molecule, has no effect on the permeability of the membrane. However, these liposomes lose integrity upon the adsorption of polycation; as a result, complexation becomes irreversible. Electroneutral and anionic surfactants with long hydrocarbon chains may accumulate in a cellular membrane owing to the oxidative degradation of unsaturated radicals in lipid molecules. This finding may be used in the design of polymeric therapeutic means specifically interacting with damaged cells.     

Complexes of anionic liposomes and a cationic polymer: Composition,structure, and characteristics

Adsorption of the synthetic polycation poly-N-ethyl-4-vinylpyridinium bromide (PEP) on the surface of bilayered lipid vesicles (liposomes) is studied. Two types of liposomes are used: (i) traditional two-component liposomes formed from neutral phosphatidylcholine (PC) and anionic diphosphatidylglycerol (cardiolipin, CL²⁻) and (ii) PC/CL²⁻ anionic liposomes with the built-in nonionogenic surfactant poly(ethyleneglycol) cetyl ether with a degree of polymerization of 20 (Brij-58). PEP is quantitatively linked with both types of liposomes; this process is electrostatic in character and fully reversible. The formation of a poly(ethylene glycol) layer on liposomal membrane decreases the stability of polycation-liposome complexes in aqueous salt solutions. Adsorption of PEP on the surface of PC/CL²⁻ liposomes is accompanied by their aggregation; PC/CL²⁻/Brij liposomes do not aggregates, even during complete neutralization of their charge by the adsorbed PEP. DSC measurements showed that the adsorption of the polycation is accompanied by microphase separation in the liposomal membrane: formation of domains, which are composed primarily of CL²⁻ molecules and linked to the complex with PEP, and regions, where electroneutral lipids are primarily concentrated. With the use of a spin probe, the packing density of bilayers (their microviscosity) is estimated, and a preferential localization of the probe at the boundaries of lipid domains in the membrane based on PC/CL²⁻/Brij liposomes is proposed. The causes of the aggregative stability of three-component PC/CL²⁻/Brij liposomes are described, and the structure of the prepared polymer-liposome complexes is discussed.     

Effects of lysozyme and bovine serum albumin on membrane characteristics of dipalmitoylphosphatidylglycerol liposomes

The effects of adsorption of two kinds of proteins on the membrane characteristics of liposomes were examined at pH 7.4 in terms of adsorption amounts of proteins on liposomes, penetrations of proteins into liposomal bilayer membranes, phase transition temperature, microviscosity and permeability of liposomal bilayer membranes, using positively charged lysozyme (LSZ) and negatively charged bovine serum albumin (BSA) as proteins and negatively charged L-alpha-dipalmitoylphosphatidylglycerol (DPPG) liposomes. The saturated adsorption amount of LSZ was 720 g per mol of liposomal DPPG, while that of BSA was 44 g per mol of liposomal DPPG. The penetration of LSZ into DPPG lipid membranes was greater than that of BSA. The microviscosity in the hydrophobic region of liposomal bilayer membranes increased due to adsorption (penetration) of LSZ or BSA, while the permeability of liposomal bilayer membranes increased. The gel-liquid crystalline phase transition temperature of liposomal bilayer membranes was not affected by adsorption of LSZ or BSA, while the DSC peak area (heat of phase transition) decreased with increasing adsorption amount of LSZ or BSA. It is suggested that boundary DPPG makes no contribution to the phase transition and that boundary DPPG and bulk DPPG are in the phase-separated state, thereby increasing the permeability of liposomal bilayer membranes through adsorption of LSZ or BSA. A possible schematic model for the adsorption of LSZ or BSA on DPPG liposomes was proposed.     

Effect of adsorption of bovine serum albumin on liposomal membrane characteristics

The effect of adsorption of bovine serum albumin (BSA) on the membrane characteristics of liposomes at pH 7.4 was examined in terms of zeta potential, micropolarity, microfluidity and permeability of liposomal bilayer membranes, where negatively charged L-alpha-dipalmitoylphosphatidylglycerol (DPPG)/L-alpha-dipalmitoylphosphatidylcholine (DPPC), negatively charged dicetylphosphate (DCP)/DPPC and positively charged stearylamine (SA)/DPPC mixed liposomes were used. BSA with negative charges adsorbed on negatively charged DPPG/DPPC mixed liposomes but did not adsorb on negatively charged DCP/DPPC and positively charged SA/DPPC mixed liposomes. Furthermore, the adsorption amount of BSA on the mixed DPPG/DPPC liposomes increased with increasing the mole fraction of DPPG in spite of a possible electrostatic repulsion between BSA and DPPG. Thus, the adsorption of BSA on liposomes was likely to be related to the hydrophobic interaction between BSA and liposomes. The microfluidity of liposomal bilayer membranes near the bilayer center decreased by the adsorption of BSA, while the permeability of liposomal bilayer membranes increased by the adsorption of BSA on liposomes. These results are considered to be due to that the adsorption of BSA brings about a phase separation in liposomes and that a temporary gap is consequently formed in the liposomal bilayer membranes, thereby the permeability of liposomal bilayer membranes increases by the adsorption of BSA.     

Migration of a cationic polymer between lipid vesicles

The adsorption of a synthetic polycation, poly(N-ethyl-4-vinylpyridinium bromide) (PEVP), on the surface of bilayer lipid vesicles (liposomes) and the migration of adsorbed macromolecules between the liposomes are studied. Liposomes of three types are used, including (1) traditional two-component liposomes composed of neutral phosphatidylcholine (PC) and anionic cardiolipin (CL); (2) three-component liposomes consisting of PC, CL, and cationic dicetyldimethylammonium bromide (DCMAB); and (3) anionic PC/CL liposomes with a nonionic surfactant, poly(ethylene oxide)-cetyl alcohol ether (Briij 58), incorporated into their bilayers. The adsorption of PEVP on the surface of PC/CL liposomes is accompanied by their aggregation. Using the fluorescence method, it is shown that the units (segments) of the polycation undergo partial redistribution between the liposomes inside the aggregates formed from PC/CL liposomes (with and without a fluorescent label) and PEVP. On the contrary, three-component PC/CL/DCMAB and PC/CL/Briij liposomes are not aggregated, even with the complete neutralization of their charges by adsorbed PEVP. In both cases, the migration of PEVP molecules between individual (nonaggregated) liposomes is observed. Possible reasons for the aggregative stability of the three-component PC/CL/DCMAB and PC/CL/Briij liposomes and the mechanism of interliposome migration of PEVP in such systems are discussed.     

Complexation of polycations to anionic liposomes: composition and structure of the interfacial complexes

Poly(N-ethyl-4-vinylpyridinium bromide) (a polycation with a degree of polymerization of 1100) was adsorbed onto liposomes composed of egg lecithin with a 0.05-0.20 molar fraction (nu) of anionic headgroups provided by cardiolipin (a doubly anionic lipid). According to electrophoretic mobility data, this led to total charge neutralization of the liposomes, whereupon the liposomes adopted a positive charge as additional polymer continued to adsorb. Although the liposomes aggregated at the charge-neutralization point, they disassembled into individual liposomes after becoming positively charged. The degree of polymer adsorption was shown to reach a limit. Thus, by measuring the free polymer content in a liposome suspension, it was possible to determine the polymer concentration at which the liposome surface became saturated with polymer. Beyond this point, an electrostatic/steric barrier at the surface suppressed further adsorption. Dynamic light scattering studies of liposomes with and without adsorbed polymer allowed calculation of the polymer film thickness which ranged from 22 to 35 nm as the molar fraction of cardiolipin (nu) increased from 0.05 to 0.20. The greater the content on the anionic lipid in the bilayer, the thicker the polymer film. The maximum number of polymer molecules adsorbed onto the liposomes was estimated: 1-2 molecules for nu = 0.05; 3 molecules for nu = 0.1; 4- molecules for nu = 0.15; and 6 molecules for nu = 0.2. The polymer appears to lie on the liposome surface, rather than embedding into the bilayer, because addition of NaCl easily dislodges the polymer from the liposome into the bulk water.     

Effects of incorporation of various amphiphiles into recipient liposome membranes on inter-membrane protein transfer.

To obtain information about the factors governing spontaneous inter-membrane protein transfer, we examined the effects of incorporation of various amphiphilic compounds in dimyristoylphosphatidylcholine (DMPC) liposomes on protein transfer from influenza virus-infected cells to the liposomes, and analyzed the physical properties of these liposome membranes. The incorporation of amphiphilic compounds, negatively charged dicetylphosphate (DCP), dipalmitoylphosphatidylserine (DPPS) or positively charged dimethyldipalmitoylammonium (DMDPA), into DMPC liposomal membranes enhanced protein transfer. The liposomes containing DCP, DPPS or DMDPA were unaffected by osmotic shock caused by external addition of glucose, suggesting a decrease in lipid packing in the liposomal membranes. Furthermore, calorimetric study of these liposomes showed that a phase separation occurred partially in the liposomal membranes. Accordingly, the membranes of DMPC liposomes containing DCP, DPPS and DMDPA should be distorted due to the coexistence of two phases, gel and liquid crystalline, in the membranes. Consequently, the membrane distortion could be responsible for the enhancement effects of the amphiphiles on the inter-membrane protein transfer from influenza virus-infected cells to the liposomes.     

Stability of anionic liposome-cationic polymer complexes in water-salt media

Laser microelectrophoresis, dynamic light scattering, and fluorescence and UV spectroscopy are employed to study poly-N-ethyl-4-vinylpyridinium bromide adsorption on the surface of bilayer lipid vesicles (liposomes) formed from mixtures of anionic phosphatidyl serine and electroneutral phosphatidylcholine. It is established that polycation adsorption is accompanied by the neutralization of charges on liposomes and their aggregation. The subsequent addition of a low-molecular-weight salt (NaCl) solution to suspensions of complexes causes them to dissociate into their initial components, while the stability of the complexes with respect to the salt action increases with the fraction of the anionic lipid in the liposome membranes. The data obtained are interpreted from the position of the formation-disintegration of a molecular capacitor, the charge of which is generated by spatially separated anionic lipids located in the bilayer membrane and cationic units of the adsorbed polyamine.     

Dynamic polarization of lipid bilayers as a special case in the electrochemistry of liquid/liquid interfaces

Dynamically obtained current/voltage curves of bilayer lipid membranes partitioning a solution of lipophilic ions are compared with the results of the type expected in a voltammetry experiment involving ionic transport across a liquid/liquid interface. Lipophilic ions yielding “voltammograms” analogous to reversible and irreversible voltammograms (in conventional electrochemical systems) are reported. Also reported are examples of ions which yield what may be analogous to a masked response, a phenomenon known in the literature of liquid/liquid interfaces. Although the behavior of the two systems is similar, there exist differences in the interpretation of the voltammograms and suggestions are offered for an energetic and mechanistic interpretation of the membrane voltammogram.     

Chromatographic purification steps involved in the synthesis of prodrug-liposome-antibody-complexes

Summary The cytostatic effect of the widely used antitumor drug 1-β-D-arabinofuranosyl cytosine (ara C) can be improved by its chemical derivatization to lipophilic prodrugs. We have incorporated these prodrugs together with lipophilic biotin derivatives into membranes of unilamellar liposomes. Monoclonal antibodies were coupled to the biotin residues of the liposomes via avidin-biotin complexation resulting in prodrug-liposome-antibody complexes whichin vitro preferably bind to cells selectively recognized by the immobilized antibodies. The results open a promising way of drug targeting. The components and liposomal derivatives used for the stepwise preparation of the prodrug-liposome antibody complex are purified by means of preparative liquid chromatography. Lipophilic membrane components are chromatographed on silica gel, antibodies on hydroxylapatite and liposomal derivatives on Ultrogel AcA 22 columns. Concentration and desalting are achieved by ultrafiltration. The purification process can be quantitatively pursued by labelling with radioactive components.     

Influence of macromolecule adsorption during filtration of a membrane bioreactor mixed liquor suspension

The aim of this study was to quantify the specific effect of adsorption on membrane fouling during filtration of a membrane bioreactor (MBR) mixed liquor suspension. Adsorption experiments were performed on well-defined protein solutions (β-lactoglobulin solutions) to provide reference results and compare them to those obtained during the filtration of MBR suspensions (raw suspension and settled suspension). Two different methods were used to quantify the role of adsorption in membrane fouling: a “static” method in which membranes were immersed in the biological suspension and a “dynamic” method supposing that the resistance due to adsorption is an irreversible phenomenon that remains after filtration and back-washing. It was shown for the two types of suspensions that (i) due to limited diffusion, the dynamic method appears to be more adapted than the static method; (ii) adsorption is a rapid fouling phenomenon that induces irreversible resistance and that, in frontal mode takes place at the beginning of the operation; (iii) the adsorption phenomenon shows specific hydraulic resistance of the same order of magnitude as the clean membrane resistance; (iv) other phenomena, i.e. progressive pore clogging, can also take place though subcritical hydrodynamic conditions.     

Flat hydrogel substrate for atomic force microscopy to observe liposomes and lipid membranes

In order to avoid denaturation of biomolecules due to strong adsorption on solid surfaces, a soft substrate has to be used for atomic force microscopy (AFM) observation. We propose a hydrophilic agarose gel surface as a soft substrate for AFM to observe liposomes and lipid membranes. Although our simple method does not require any delicate control at the molecular level, an agarose gel surface can be simply flattened to 0.3 nm in roughness using an atomically flat solid surface during gelation. The AFM images revealed that liposomes were unruptured on the gel surface at low liposome density, whereas an unruptured state was difficult to obtain on a solid surface like mica. This indicates that the weak interaction between the liposome and the soft surface inhibits the liposome from rupturing, and also that the surface rougher than the solid surface prevents lateral diffusion of the liposomes along the surface to be fused. Increasing the liposome density resulted in a lipid membrane at various thicknesses forming on the hydrogel surface by the fusion and rupture of liposomes. Using the soft substrate, it can be expected to promote investigations of structures and functions of biomolecules at the nanometer scale under physiological conditions with AFM.     

Comparative study of the dark and light-induced toxicity of lipofuscin granules from human retinal pigment epithelium and their chromophore A2E on the cardiolipin liposome model

The damaging effect of lipofuscin granules from the human retinal pigment epithelium and fluorophore A2E was studied on models of calcein- and ascorbate-loaded cardiolipin liposomes and outer segments of the bovine eye photoreceptor cells in dark and under visible light irradiation. In dark fluorophore A2E induces the release of calcein from calcein-loaded liposomes and reduces the lifetime of the artificial bilayer lipid membrane prepared from dioleyl phosphatidilcholine. A similar detergent-like action A2E exhibits towards ascorbate-loaded liposomes, significantly accelerating the release of ascorbate in dark. In the presence of A2E, irradiation with the full visible light (390?C700 nm) stimulates both the release of ascorbate from liposomes and accelerates the destruction of the bilayer lipid membrane. Retinal pigment epithelium lipofuscin granules also accelerate the release of ascorbate from ascorbate-loaded liposomes under visible light irradiation; the blue light (457.9 nm) was twice as more efficient as the green light (514.5 nm). The preliminary irradiation of A2E with the visible light decreases its detergent-like action on the cardiolipin liposomal membranes under the dark conditions and the photosensitizing effect on the lipid peroxidation of the outer segments of photoreceptor cells. Unlike A2E, the visible light irradiation of a suspension of lipofuscin granules under similar conditions does not noticeably decrease their sensitizing activity towards lipid peroxidation. It is assumed that the phototoxicity of retinal pigment epithelium lipofuscin granules is related not only to A2E in their composition, but depends mainly on the content of other photosensitizers (chromophores) in the granules.     

Contrasting behavior of zwitterionic and cationic polymers bound to anionic liposomes

Zwitterionic polymers were prepared by quaternizing polyvinylpyridine (DP = 1100) with bromoacids (Br(CH2)nCOOH, where n = 1, 2, 3, and 5). The resulting polymers were then added to unilamellar liposomes composed of egg lecithin or dipalmitoylphosphatidylcholine admixed with 20 mol % of cardiolipin (a phospholipid with two negative charges). These systems were compared (along with polyethylvinylpyridinium chloride, a polycation) by light scattering, electrophoretic mobility, fluorescence, and high-sensitivity differential scanning calorimetry. The external zwitterionic polymers induce no flip-flop of cardiolipin from the inner leaflet to the outer leaflet as does the polycation. Aside from this similarity, the four zwitterionic polymers all behave differently from each other toward the anionic liposomes: (a) For n = 1, there is no detectable interaction between the polymer and the liposomes. (b) For n = 2, electrostatic attraction induces polymer-liposome association (reversed by the addition of NaCl) that maintains the original negative charge on the liposome. Aggregation of the liposomes accompanies polymer adsorption. (c) For n = 3, electrostatic binding also occurs along with aggregation. However, the binding is so strong that NaCl is unable to induce polymer/liposome dissociation. (d) For n = 5, there is polymer binding and NaCl-promoted dissociation but no substantial aggregation. These differences among the closely related polymers are discussed and analyzed in molecular terms.     

The physicochemical/thermodynamic balance of advanced drug liposomal delivery systems

The aim of this work is to study the morphological characteristics via fractal analysis and the alterations of the thermotropic behavior of dipalmitoylphosphatidylcholine (DPPC) liposomes, caused by the incorporation of cholesterol, poly(amidoamine) (PAMAM) dendrimer, and MPOx (poly(2-methyl-2-oxazoline)-grad-poly(2-phenyl-2-oxazoline)) gradient block copolymer (9:1 molar ratio). A gamut of light scattering techniques and differential scanning calorimetry were used in order to extract information on the morphological (in different dispersion media) and thermodynamic characteristics of liposomal drug nanocarriers, respectively. The vesicles’ structure of liposomes has a different thermodynamic content, which corresponds to a different thermotropic behavior, in comparison to pure lipid bilayers. The observed metastable phase only for DPPC liposomes has been considered as a “physical impurity”, which leads to “physical incompatibility” and consequently promotes the aggregation of DPPC liposomes in aqueous media. The incorporation of biomaterials such as PAMAM G4 and MPOx, caused alterations in the thermotropic behavior of DPPC liposomes affecting only the main transition specific enthalpy ΔH. All the other calorimetric parameters remained unaltered. These findings supported the hypothesis that the exceptional stability and transition cooperativity of the chimeric liposomal membrane might be due to the reduction of the vesicle size with the smaller membrane curvature that is indicated by the fractal dimensionality of the system. In conclusion, the results from the thermal analysis of the liposomal systems were in line with the picture of their structural characteristics, as indicated by the interplay between physicochemical and thermodynamical parameters, which determines their fractal morphology.     

Discrete Arrays of Liquid‐Crystal‐Supported Proteolipid Monolayers as Phantom Cell Surfaces

The phospholipid bilayers of living cell membranes exist almost universally in a liquid state. This enables motion and spatial reorganization of membrane components on multiple length scales, which is an essential feature of many biological processes. There is great interest in the development of molecularly defined interfaces between synthetic materials and living cells. To this end, there is a need for solid substrate materials that can be derivatized with fluid, membrane‐like interfaces. Herein, we describe array fabrication of discrete liquid‐crystal areas supporting phospholipid monolayer membranes, and characterize the interactions with several different membrane surface proteins [avidin series, cholera toxin, green fluorescent protein (GFP), intercellular adhesion molecule (ICAM) and major histocompatibility complex (MHC)]. Three different linkage strategies (biotin, nickel chelating lipids complexing with histidine, and the choleratoxin binding unit (CTB) associating with G_M1 are evaluated. Additionally, experiments with live immunological T cells forming active synapses at the interface exhibit the specific nature of the surface.     

Effects of poly(ethylene glycol) (PEG) chain length of PEG-lipid on the permeability of liposomal bilayer membranes

The effects of poly(ethylene glycol) (PEG) chain length of PEG-lipid on the membrane characteristics of liposomes were investigated by differential scanning calorimetry (DSC), freeze-fracture electron microscopy (FFEM), fluorescence polarization measurement and permeability measurement using carboxyfluorescein (CF). PEG-liposomes were prepared from mixtures of dipalmitoyl phosphatidylcholine (DPPC) and distearoyl phosphatidylethanolamines with covalently attached PEG molecular weights of 1000, 2000, 3000 and 5000 (DSPE-PEG). DSC and FFEM results showed that the addition of DSPE-PEG to DPPC in the preparation of liposomes caused the lateral phase separation both in the gel and liquid-crystalline states. The fluidity in the hydrocarbon region of liposomal bilayer membranes was not significantly changed by the addition of DSPE-PEG, while that in the interfacial region was markedly increased. From these results, it was anticipated that the CF leakage from PEG-liposomes is accelerated compared with DPPC liposomes. However, CF leakage from liposomes containing DSPE-PEG with a 0.060 mol fraction was depressed compared with regular liposomes, and the leakage decreased with increasing PEG chain length. Furthermore, the CF leakage from liposomes containing DSPE-PEG with a 0.145 mol fraction was slightly increased compared with that of liposomes containing DSPE-PEG with a 0.060 mol fraction. It is suggested that the solute permeability from the PEG-liposomes was affected by not only properties of the liposomal bilayer membranes such as phase transition temperature, phase separation and membrane fluidity, but also the PEG chain of the liposomal surface.     

Membrane properties of cationic liposomes composed of dipalmitoylphosphatidylcholine and dipalmityldimethylammonium bromide

Cationic liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and dipalmityldimethylammmonium bromide (DPAB) were prepared by the Bangham method and the effect of DPAB on the membrane properties was examined in terms of liposomal shape, particle size, trapping efficiency, surface potential and dispersibility. The dispersibility of the mixed DPPC/DPAB liposomes (the mole fraction of DPAB (X_DPAB)  0.05) was excellent and the dispersibility was maintained for 6 months, since the zeta-potential of the mixed liposomes was approximately +40 mV. The trapping efficiency of the mixed DPPC/DPAB liposomes (X_DPAB = 0.05) was 10 times greater than that of the DPPC liposomes, and the value was largest among the mixed liposomes (X_DPAB = 0–1.0). Freeze-fracture electron micrographs indicated that the shape of the mixed DPPC/DPAB liposomes (X_DPAB = 0.05) was that of large unilamellar vesicles (LUVs) with a diameter of approximately 2 μm, while the shape of the DPPC liposomes was that of multilamellar vesicles (MLVs). The mixed liposomes had, therefore, a high trapping efficiency. Furthermore, the shape of the mixed DPPC/DPAB liposomes (X_DPAB = 0.75) was also that of LUVs with a diameter of approximately 2 μm and these had a high trapping efficiency. Whereas, the particle size (500 nm) of the mixed DPPC/DPAB liposomes (X_DPAB = 0.25) was smaller than that of the former and had the minimum trapping efficiency. The phase transition temperature of the liposomal bilayer membranes indicated a maximum value at 0.25–0.30 mole fractions of DPAB. These facts were considered to be due to the fact that DPPC and DPAB, whose molar ratio was 7.5:2.5, were tightly packed in the liposomal bilayer membranes and that the curvature of the liposomal particle was resultantly large. Nevertheless, LUVs having a high trapping efficiency were easily obtained by mixing a small amount of DPAB with the DPPC.