Multicomponent cationic lipid-DNA complex formation: role of lipid mixing

Multicomponent cationic lipid-DNA complexes (lipoplexes) were prepared by adding linear DNA to mixed lipid dispersions containing two populations of binary cationic liposomes and characterized by means of small angle X-ray scattering (SAXS). Four kinds of cationic liposomes were used. The first binary lipid mixture was made of the cationic lipid (3'[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol) and the neutral helper lipid dioleoylphosphocholine (DOPC) (DC-Chol/DOPC liposomes), the second one of the cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the neutral dioleoylphosphatidylethanolamine (DOPE) (DOTAP/DOPE liposomes), the third one of DC-Chol and DOPE (DC-Chol/DOPE liposomes), and the fourth one of DOTAP and DOPC (DOTAP/DOPC liposomes). Upon DNA-induced fusion of liposomes, large lipid mixing at the molecular level occurs. As a result, highly organized mixed lipoplexes spontaneously form with membrane properties intermediate between those of starting liposomes. By varying the composition of lipid dispersions, different DNA packing density regimes can also be achieved. Furthermore, occurring lipid mixing was found to induce hexagonal to lamellar phase transition in DOTAP/DOPE membranes. Molecular mechanisms underlying experimental findings are discussed.     

Structural evolution of environmentally responsive cationic liposome-DNA complexes with a reducible lipid linker

Environmentally responsive materials (i.e., materials that respond to changes in their environment with a change in their properties or structure) are attracting increasing amounts of interest. We recently designed and synthesized a series of cleavable multivalent lipids (CMVLn, with n = 2-5 being the number of positive headgroup charges at full protonation) with a disulfide bond in the linker between their cationic headgroup and hydrophobic tails. The self-assembled complexes of the CMVLs and DNA are a prototypical environmentally responsive material, undergoing extensive structural rearrangement when exposed to reducing agents. We investigated the structural evolution of CMVL-DNA complexes at varied complex composition, temperature, and incubation time using small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS). A related lipid with a stable linker, TMVL4, was used as a control. In a nonreducing environment, CMVL-DNA complexes form the lamellar (L(α)(C)) phase, with DNA rods sandwiched between lipid bilayers. However, new self-assembled phases form when the disulfide linker is cleaved by dithiothreitol or the biologically relevant reducing agent glutathione. The released DNA and cleaved CMVL headgroups form a loosely organized phase, giving rise to a characteristic broad SAXS correlation profile. CMVLs with high headgroup charge also form condensed DNA bundles. Intriguingly, the cleaved hydrophobic tails of the CMVLs reassemble into tilted chain-ordered L(β') phases upon incubation at physiological temperature (37 °C), as indicated by characteristic WAXS peaks. X-ray scattering further reveals that two of the three phases (L(βF), L(βL), and L(βI)) constituting the L(β') phase coexist in these samples. The described system may have applications in lipid-based nanotechnologies.     

Silicon supported lipid-DNA thin film structures at varying temperature studied by energy dispersive X-ray diffraction and neutron reflectivity

Non-viral gene transfection by means of lipid-based nanosystems, such as solid supported lipid assemblies, is often limited due to their lack of stability and the consequent loss of efficiency. Therefore not only a detailed thermo-lyotropic study of these DNA-lipid complexes is necessary to understand their interaction mechanisms, but it can also be considered as a first step in conceiving and developing new transfection biosystems. The aim of our study is a structural characterization of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)-dimethyl-dioctadecyl-ammonium bromide (DDAB)-DNA complex at varying temperature using the energy dispersive X-ray diffraction (EDXD) and neutron reflectivity (NR) techniques. We have shown the formation of a novel thermo-lyotropic structure of DOPC/DDAB thin film self-organized in multi-lamellar planes on (100)-oriented silicon support by spin coating, thus enlightening its ability to include DNA strands. Our NR measurements indicate that the DOPC/DDAB/DNA complex forms temperature-dependent structures. At 65°C and relative humidity of 100% DNA fragments are buried between single lamellar leaflets constituting the hydrocarbon core of the lipid bilayers. This finding supports the consistency of the hydrophobic interaction model, which implies that the coupling between lipid tails and hypo-hydrated DNA single strands could be the driving force of DNA-lipid complexation. Upon cooling to 25°C, EDXD analysis points out that full-hydrated DOPC-DDAB-DNA can switch in a different metastable complex supposed to be driven by lipid heads-DNA electrostatic interaction. Thermotropic response analysis also clarifies that DOPC has a pivotal role in promoting the formation of our observed thermophylic silicon supported lipids-DNA assembly.     

Milk membrane lipid vesicle structures studied with Cryo-TEM

Milk fat globule membrane (MFGM) lipids have been studied in the presence and absence of proteins β-lactoglobulin and β-casein. The aim of this study was to relate the self-assembly structure, e.g. vesicles, formed in aqueous dispersions of MFGM lipids to the lipid composition, electrolyte composition as well as the effect of added milk proteins, i.e. β-lactoglobulin and β-casein. For this purpose, vesicles of phospholipid mixtures, containing dioleoylphosphatidylcholine (DOPC), sphingomyelin (SM), dioleoylphosphatidylethanolamine (DOPE), phosphatidylinositol (PI) and dioleoylphosphatidylserine (DOPS) at composition corresponding to that of the MFGM, were prepared by extrusion. The morphology of the formed structures of different sample compositions was studied with cryogenic transmission electron microscopy (Cryo-TEM). Mixtures of membrane lipid with a composition (e.g. 80% DOPE, 12% DOPC and 8% SM) that at high lipid content give liquid crystalline phases at the boundary of lamellar to reversed hexagonal phase rather formed microtubular structures than vesicles at high water content. A large proportion of multilamellar vesicles is formed in buffer and divalent salts than in pure water. A small increase in the interlayer spacing of the multilamellar vesicle was observed in the presence of β-casein.     

Block liposomes from curvature-stabilizing lipids: connected nanotubes, -rods, or -spheres

We report on the discovery of block liposomes, a new class of chain-melted (liquid) vesicles, with membranes comprised of mixtures of the membrane-curvature-stabilizing multivalent lipid MVLBG2 of colossal charge +16 e and neutral 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). In a narrow MVLBG2 composition range (8-10 mol%), cryo-TEM revealed block liposomes consisting of distinctly shaped, yet connected, nanoscale spheres, pears, tubes, or rods. Unlike typical liposome systems, where spherical vesicles, tubular vesicles, and cylindrical micelles are separated on the macroscopic scale, within a block liposome, shapes are separated on the nanometer scale. Diblock (pear-tube) and triblock (pear-tube-pear) liposomes contain nanotubes with inner lumen diameter of 10-50 nm. Diblock (sphere-rod) liposomes were found to contain micellar nanorods approximately 4 nm in diameter and several micrometers in length, analogous to cytoskeletal filaments of eukaryotic cells. Block liposomes may find a range of applications in chemical and nucleic acid delivery and as building blocks in the design of templates for hierarchical structures.     

Thermotropic and lyotropic phase properties of glycolipid diastereomers: role of headgroup and interfacial interactions in determining phase behaviour

Recent advances in the area of glycobiology have been paralleled by progress in our understanding of the physical properties of glycoglycerolipids (GGLs). These advances have been accelerated by interest in the new found roles of these simple glycolipids in nature, by advances in synthetic procedures, and by an interest in the technological application of a group of amphiphiles with unique physical and chemical properties. Here, we consider the phase properties of some GGL/water systems containing either a single hexopyranoside or pentopyranoside headgroup. Recent calorimetric and X-ray diffraction measurements of some GGL diastereomers suggest that both headgroup and interfacial hydration play a major role in determining both lyotropism and mesomorphic phase properties as the chemical structure of the lipid headgroup, interface and hydrocarbon chains are systematically altered. For GGLs of a given chain length, interactions between the headgroup/interface and water determine whether or not a highly ordered, lamellar crystalline phase is formed, the number of such phases and their rate of formation and, in some cases, the nature of the molecular packing of those phases. In the liquid crystalline phases, the hydrocarbon chains determine the area per molecule in the lamellar liquid crystalline phase, but it is the cross-sectional area of the hydrated headgroup and the penetration of water into the interface which determines the nature of the non-lamellar phases, probably through small changes in interfacial geometry as the lateral stresses in the headgroup region increase.     

Activity of synthetic ion channels is influenced by cation-pi interactions with phospholipid headgroups

A suite of synthetic hydraphile ion channels has been used to probe the possibility of cation-pi interactions between the channel and the phospholipid bilayer. The hydraphiles selected for this study contained either no sidearm, aliphatic sidearms or aromatic sidearms that varied in electron-richness. An ion selective electrode (ISE) method was used to evaluate the ion transport ability of these hydraphiles across synthetic bilayers. Transport was dependent on sidearm identity. Ion transport activity for the aromatic sidechained compounds was greatest when the sidearms were electron rich and vesicles were prepared from 100% DOPC (trimethylammonium cation headgroup, overall neutral). When the lipid headgroups were made more negative by changing the composition from DOPC to 70 : 30 (w/w) DOPC : DOPA, transport by the aromatic-sidechained channels was reduced. Fluorescence studies showed that when the lipid composition changed, the headgroups experienced a different polarity, suggesting reorientation. The data are in accord with a stabilizing cation-pi interaction between the aromatic sidearm of the hydraphile channel and the ammonium phospholipid headgroup.     

Anion transport properties of amine and amide-sidechained peptides are affected by charge and phospholipid composition

Four synthetic anion transporters (SATs) having the general formula (n-C(18)H(37))(2)N-COCH(2)OCH(2)CO-(Gly)(3)Pro-Lys(epsilon-N-R)-(Gly)(2)-O-n-C(7)H(15) were prepared and studied. The group R was Cbz, H (TFA salt), t-Boc, and dansyl in peptides 1, 2, 3, and 4 respectively. The glutamine analog (GGGPQAG sequence) was also included. A dansyl-substituted fluorescent SAT was used to probe peptide insertion; the dansyl sidechain resides in an environment near the bilayer's midpolar regime. When the lysine sidechain was free or protected amine, little effect was noted on final Cl(-) transport rate in DOPC : DOPA (7 : 3) liposomes. This stands in contrast to the significant retardation of transport previously observed when a negative glutamate residue was present in the peptide sequence. It was also found that Cl(-) release from liposomes depended on the phospholipid composition of the vesicles. Chloride transport diminished significantly for the free lysine containing SAT, 2, when the lipid was altered from DOPC : DOPA to pure DOPC. Amide-sidechained SATs 1 and 5 showed a relatively small decrease in Cl(-) transport. The effect of lipid composition on Cl(-) transport was explained by differences in electrostatic interaction between amino acid sidechain and lipid headgroup, which was modeled by computation.     

Remodeling of ordered membrane domains by GPI-anchored intestinal alkaline phosphatase

Glycosylphosphatidyl-inositol (GPI)-anchored proteins preferentially localize in the most ordered regions of the cell plasma membrane. Acyl and alkyl chain composition of GPI anchors influence the association with the ordered domains. This suggests that, conversely, changes in the fluid and in the ordered domains lipid composition affect the interaction of GPI-anchored proteins with membrane microdomains. Validity of this hypothesis was examined by investigating the spontaneous insertion of the GPI-anchored intestinal alkaline phophatase (BIAP) into the solid (gel) phase domains of preformed supported membranes made of dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC), DOPC/sphingomyelin (DOPC/SM), and palmitoyloleoylphosphatidylcholine/SM (POPC/SM). Atomic force microscopy (AFM) showed that BIAP inserted in the gel phases of the three mixtures. However, changes in the lipid composition of membranes had a marked effect on the protein containing bilayer topography. Moreover, BIAP insertion was associated with a net transfer of phospholipids from the fluid to the gel (DOPC/DPPC) or from the gel to the fluid (POPC/SM) phases. For DOPC/SM bilayers, transfer of lipids was dependent on the homogeneity of the gel SM phase. The data strongly suggest that BIAP interacts with the most ordered lipid species present in the gel phases of phase-separated membranes. They also suggest that GPI-anchored proteins might contribute to the selection of their own microdomain environment.     

Enhanced transfection efficiency of multicomponent lipoplexes in the regime of optimal membrane charge density

Recently, membrane charge density of lipid membranes, sigma M, has been recognized as a universal parameter that controls the transfection efficiency of complexes made of binary cationic liposomes and DNA (binary lipoplexes). Three distinct regimes, most likely related to interactions between complexes and cells, have also been identified. The purpose of this work was to investigate the transfection efficiency behavior of multicomponent lipoplexes in the regime of optimal membrane charge density (1< sigma M < 2 x 10 (-2) e/A (2)) and compare their performance with that of binary lipoplexes usually employed for gene delivery purposes. We found remarkable differences in transfection efficiency due to lipid composition, with maximum in efficiency being obtained when multicomponent lipoplexes were used to transfect NIH 3T3 cells, while binary lipoplexes were definitely less efficient. These findings suggested that multicomponent systems are especially promising lipoplex candidates. With the aim of providing new insights into the mechanism of transfection, we investigated the structural evolution of lipoplexes when interacting with anionic (cellular) lipids by means of synchrotron small-angle X-ray diffraction (SAXD), while the extent of DNA release upon interaction with anionic lipids was measured by electrophoresis on agarose gels. Interestingly, a clear trend was found that the transfection activity increased with the number of lipid components. These results highlight the compositional properties of carrier lipid/cellular lipid mixtures as decisive factors for transfection and suggest a strategy for the rational design of superior cationic lipid carriers.     

A supramolecular polymerisation model for the structurisation of DNA–lipid complexes

Complexes formed by DNA and lipid mixtures have great interest for the assessment of self-assembling mechanisms in open biological systems. X-ray diffraction has revealed that minor alterations of the cationic/neutral lipid composition produced major alterations to the liquid crystalline structure and the hydration of the complexes. We have extended to these systems by an approach based on the identification of a fundamental repeating unit that grows according to the general principles of supramolecular polymerisation and liquid crystallinity. Structural reorganisations that optimise the electrostatic and hydrophobic compensation are enhanced by competition between the rigidity of the polyelectrolyte and the cohesion of the lipid assembly. The non-hydrated hexagonal structure revealed by X-ray examination is represented by a dendritic-type supramolecular polymerisation of DNA units decorated by the aliphatic tails of dissociated liposomes. An increase in the cationic/neutral lipid component ratio enhances the stability of planar bilayers, favouring the formation of the partly hydrated lamellar structure revealed by X-ray diffraction.     

Cholesterol slows down the lateral mobility of an oxidized phospholipid in a supported lipid bilayer

We investigated the mobility and phase-partitioning of the fluorescent oxidized phospholipid analogue 1-palmitoyl-2-glutaroyl-sn-glycero-3-phospho-N-Alexa647-ethanolamine (PGPE-Alexa647) in supported lipid bilayers. Compared to the conventional phospholipid dihexadecanoylphosphoethanolamine (DHPE)-Bodipy we found consistently higher diffusion constants. The effect became dramatic when immobile obstacles were inserted into the bilayer, which essentially blocked the diffusion of DHPE-Bodipy but hardly influenced the movements of PGPE-Alexa647. In a supported lipid bilayer made of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), the differences in probe mobility leveled off with increasing cholesterol content. Using coarse-grained molecular dynamics simulations, we could ascribe this effect to increased interactions between the oxidized phospholipid and the membrane matrix, concomitant with a translation in the headgroup position of the oxidized phospholipid: at zero cholesterol content, its headgroup is shifted to the outside of the DOPC headgroup region, whereas increasing cholesterol concentrations pulls the headgroup into the bilayer plane.     

Effect of PIP2 on Bilayer Structure and Phase Behavior of DOPC: An X‐ray Scattering Study

Phosphatidylinositol 4,5‐bis‐phosphate (PIP₂) is an important lipid in regulation of several cellular processes, particularly membrane fusion. We use X‐ray diffraction from solid‐supported multilamellar 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC)/PIP₂ samples to study changes in bilayer structure and the lyotropic phase behavior induced by physiologically relevant concentrations of PIP₂. Electron‐density profiles reconstructed from X‐ray reflectivity measurements indicate that PIP₂ strongly affects structural parameters such as lipid head‐group width, bilayer thickness, and lamellar repeat spacing of DOPC bilayer stacks. In addition, at lower degrees of hydration, a few molar per cent of PIP₂ facilitates stalk‐phase formation and also leads to formation of a hexagonal phase, which is not observed in pure DOPC. These results indicate that the role of PIP₂ in membrane fusion could be, in part, due to its effect on the properties of the lipid bilayer matrix. Furthermore, coexistence of two lamellar phases with different lattice constants is observed in single‐component PIP₂ samples.     

Profiling Metal Oxides with Lipids: Magnetic Liposomal Nanoparticles Displaying DNA and Proteins

Metal oxides include many important materials with various surface properties. For biomedical and analytical applications, it is desirable to engineer their biocompatible interfaces. Herein, a phosphocholine liposome (DOPC) and its headgroup dipole flipped counterpart (DOCP) were mixed with ten common oxides. Using the calcein leakage assay, cryo‐TEM, and ζ‐potential measurement, these oxides were grouped into three types. The type 1 oxides (Fe₃O₄, TiO₂, ZrO₂, Y₂O₃, ITO, In₂O₃, and Mn₂O₃) form supported bilayers only with DOCP. Type 2 (SiO₂) forms supported bilayers only with DOPC; type 3 (ZnO and NiO) are cationic and damage lipid membranes. Magnetic Fe₃O₄ nanoparticles were further studied for conjugation of fluorophores, proteins, and DNA to the supported DOCP bilayers via lipid headgroup labeling, covalent linking, or lipid insertion. Delivery of the conjugates to cells and selective DNA hybridization were demonstrated. This work provides a general solution for coating the type 1 oxides with a simple mixing in water, facilitating applications in biosensing, separation, and nanomedicine.     

Headgroup hydration and mobility of DOTAP/DOPC bilayers: a fluorescence solvent relaxation study

The biophysical properties of liposome surfaces are critical for interactions between lipid aggregates and macromolecules. Liposomes formed from cationic lipids, commonly used to deliver genes into cells in vitro and in vivo, are an example of such a system. We apply the fluorescence solvent relaxation technique to study the structure and dynamics of fully hydrated liquid crystalline lipid bilayers composed of mixtures of cationic dioleoyltrimethylammoniumpropane (DOTAP) and neutral dioleoylphosphatidylcholine (DOPC). Using three different naphthalene derivatives as fluorescent dyes (Patman, Laurdan and Prodan) allowed different parts of the headgroup region to be probed. Wavelength-dependent parallax quenching measurements resulted in the precise determination of Laurdan and Patman locations within the DOPC bilayer. Acrylamide quenching experiments were used to examine DOTAP-induced dye relocalization. The nonmonotonic dependence of dipolar relaxation kinetics (occurring exclusively on the nanosecond time scale) on DOTAP content in the membrane was found to exhibit a maximum mean solvent relaxation time at 30 mol % of DOTAP. Up to 30 mol %, addition of DOTAP does not influence the amount of bound water at the level of the sn(1) carbonyls, but leads to an increased packing of phospholipid headgroups. Above this concentration, elevated lipid bilayer water penetration was observed.     

Structure–Function Relationships of New Lipids Designed for DNA Transfection

Cationic liposome/DNA complexes can be used as nonviral vectors for direct delivery of DNA‐based biopharmaceuticals to damaged cells and tissues. To obtain more effective and safer liposome‐based gene transfection systems, two cationic lipids with identical head groups but different chain structures are investigated with respect to their in vitro gene‐transfer activity, their cell‐damaging characteristics, and their physicochemical properties. The gene‐transfer activities of the two lipids are very different. Differential scanning calorimetry and synchrotron small‐ and wide‐angle X‐ray scattering give valuable structural insight. A subgel‐like structure with high packing density and high phase‐transition temperature from gel to liquid‐crystalline state are found for lipid 7 (N′‐2‐[(2,6‐diamino‐1‐oxohexyl)amino]ethyl‐2,N‐bis(hexadecyl)propanediamide) containing two saturated chains. Additionally, an ordered head‐group lattice based on formation of a hydrogen‐bond network is present. In contrast, lipid 8 (N′‐2‐[(2,6‐diamino‐1‐oxohexyl)amino]ethyl‐2‐hexadecyl‐N‐[(9Z)‐octadec‐9‐enyl]propanediamide) with one unsaturated and one saturated chain shows a lower phase‐transition temperature and a reduced packing density. These properties enhance incorporation of the helper lipid cholesterol needed for gene transfection. Both lipids, either pure or in mixtures with cholesterol, form lamellar phases, which are preserved after addition of DNA. However, the system separates into phases containing DNA and phases without DNA. On increasing the temperature, DNA is released and only a lipid phase without intercalated DNA strands is observed. The conversion temperatures are very different in the two systems studied. The important parameter seems to be the charge density of the lipid membranes, which is a result of different solubility of cholesterol in the two lipid membranes. Therefore, different binding affinities of the DNA to the lipid mixtures are achieved.     

Peculiarities of lateral diffusion of lipids in three-component bilayers

The lateral diffusion of lipid molecules in macroscopically oriented bilayers of mixed dioleoyl phosphatidylcholine (DOPC), egg sphingomyeline (SM), and cholesterol (CHOL) and its dependence on cholesterol concentration and temperature was studied by NMR with pulsed field gradient. The system forms a lamellar liquid crystalline (LC) phase; in a certain range of temperatures and concentrations of cholesterol the system is separated into two subphases: a disordered LC phase (l_d) enriched with DOPC, and an ordered phase (l₀) enriched with SM. These are characterized by their own lateral diffusion coefficients (LDCs), which differ from one another by a factor of 1.5–5. The dependence of the LDCs in the phases on the cholesterol concentration was analyzed. There was no clear dependence for the disordered LC phase, but we found that LDCs tend to grow in the concentration range of 15–35 mol % of CHOL. This behavior could be due to the redistribution of lipid components as the concentration of CHOL increases, eventually leading to a rise in DOPC concentration in the lo phase. In the range of liquid-phase domains, we observed no dependence of LDCs on the diffusion time typical of the restricted diffusion regime, due to spatial restraints in the system. This could be associated with the relatively large size of the domains, and with the domain capability of lateral diffusion in a surrounding continuous phase.     

Penetration and saturation of lysozyme in phospholipid bilayers

We show that the combination of X-ray reflectivity, tryptophan fluorescence spectrum, and fluorescence quenching by bromine provides a useful tool to probe the location of lysozyme in lipid bilayers. To this end, we prepare lamellar complexes composed of phospholipids and lysozyme on solid surfaces using a solution-casting method. The proteins lie spontaneously between adjacent bilayers in the complexes. The results indicate that lysozyme may penetrate into the lipid bilayers. But the penetration depth is very shallow, and the tryptophan residues do not penetrate beyond the interface between the hydrocardon core and the headgroup region of the lipid bilayer. The penetration becomes saturated when more proteins are incorporated into the lamellar complex. The excess proteins stay in the interlamellar aqueous layers.     

Fluidity modulation of phospholipid bilayers by electrolyte ions: insights from fluorescence microscopy and microslit electrokinetic experiments

Fluidity and charging of supported bilayer lipid membranes (sBLMs) prepared from 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) were studied by fluorescence recovery after photobleaching (FRAP) and microslit electrokinetic measurements at varying pH and ionic composition of the electrolyte. Measurements in neutral electrolytes (KCl, NaCl) revealed a strong correlation between the membrane fluidity and the membrane charging due to unsymmetrical water ion adsorption (OH(-) ? H(3)O(+)). The membrane fluidity significantly decreased below the isoelectric point of 3.9, suggesting a phase transition in the bilayer. The interactions of both chaotropic anions and strongly kosmotropic cations with the zwitterionic lipids were found to be related with nearly unhindered lipid mobility in the acidic pH range. While for the chaotropic anions the observed effect correlates with the increased negative net charge at low pH, no correlation was found between the changes in the membrane fluidity and charge in the presence of kosmotropic cations. We discuss the observed phenomena with respect to the interaction of the electrolyte ions with the lipid headgroup and the influence of this process on the headgroup orientation and hydration as well as on the lipid packaging.     

Efficient formation of giant liposomes through the gentle hydration of phosphatidylcholine films doped with sugar

Giant liposomes, or giant vesicles, are cell-size (approximately 5-100 microm) compartments enclosed with phospholipid bilayers, and have often been used in biological research. They are usually generated using hydration methods, "electroformation" and "gentle hydration (or natural swelling)", in which dry lamellar films of phospholipids are hydrated with aqueous solutions. In gentle hydration, however, giant liposomes are difficult to prepare from an electrostatically neutral phospholipid because lipid lamellae cannot repel each other. In this study, we demonstrate the efficient formation of giant liposomes using the gentle hydration of neutral phospholipid (dioleoyl phosphatidylcholine, DOPC) dry films doped with nonelectrolytic monosaccharides (glucose, mannose, and fructose). A mixture of DOPC and such a sugar in an organic solvent (chloroform/methanol) was evaporated to form the films, which were then hydrated with distilled water or Tris buffers containing sodium chloride. Under these conditions, giant liposomes spontaneously formed rapidly and assumed a swollen cell-sized spherical shape with low lamellarity, whereas giant liposomes from pure DOPC films had multilamellar lipid layers, miscellaneous shapes and smaller sizes. This observation indicates that giant unilamellar vesicles (GUVs) of DOPC can be obtained efficiently through the gentle hydration of sugar-containing lipid dry films because repulsion between lipid lamellae is enhanced by the osmosis induced by dissolved sugar.