Cubic, sponge, and lamellar phases in the glyceryl monooleyl ether-propylene glycol-water system

The phase behavior of 1-glyceryl monooleyl ether (GME) in mixtures of propylene glycol (PG) and water was investigated by visual inspection, polarization microscopy, small-angle X-ray diffraction, and conductance measurements. A phase diagram, based on over 200 samples of the ternary system GME-PG-water, was constructed at 20 degrees C. Without PG, GME forms a reverse micellar phase with up to 10 wt % water and a reverse hexagonal liquid-crystalline phase between 10 and 25 wt % water, a phase that can coexist with excess water. If PG is added in amounts exceeding about 10 wt %, then cubic and lamellar liquid-crystalline phases start to form. A cubic phase, belonging to space group Pn3m, can coexist with excess PG-water mixtures. If even more PG is added, then the cubic phase is transformed into a sponge phase. A lamellar phase forms at water contents between 10 and 15 wt % and with widely differing PG/GME weight ratios. We postulate that the phase behavior is caused by the fact that PG makes the interfacial region between self-assembled GME and PG-water less negatively curved, which in turn allows for the formation of the new phases. The phase behavior obtained for the GME system shows a striking similarity with the phase behavior of the corresponding system in which the GME has been replaced by the ester, 1-glycerol monooleate (GMO), differing only in one extra carbonyl oxygen. The major difference is the lower amount of water present in the GME phases, an effect that is mainly due to the more hydrophobic character of GME compared to that of GMO.     

Metal cation induced cubic phase in poly(ethylene glycol)-functionalized dioleoylphosphatidylethanolamine aqueous dispersions

Metal cations (Mn(2+) or Ca(2+)) in aqueous dispersions of mixtures of dioleoylphosphatidylethanolamine (DOPE) and poly(ethylene glycol)-functionalized DOPE (DOPE-PEG(350)) induce, above a certain amount of the PEG lipid component, a phase transition from the inverted hexagonal phase H(II) to the bicontinuous inverted cubic phase Q(224) with space group Pn3m. The process is driven by the decrease of free elastic energy due to the Gaussian curvature of the cubic phase. The structural characterization of the phase behavior over the whole explored range of DOPE-PEG/DOPE weight ratio (3-25%) is reported, focusing on the role of the metal cation in the formation of the 3D cubic lattice. This result may represent a significant progress toward a design-based approach to drug delivery.     

Aqueous phase behavior of a 1-O-phytanyl-beta-D-xyloside/water system. Glycolipid-based bicontinuous cubic phases of crystallographic space groups Pn3m and Ia3d

Temperature- and concentration-dependent aqueous phase diagram of a novel alkylglycoside, 1-O-phytanyl-beta-D-xyloside (beta-Xyl(Phyt)), was studied using small-angle X-ray scattering, polarizing optical microscopy, and differential scanning calorimetry. The phases found in this system include an Lc phase, an Lalpha phase, an HII phase, two inverted cubic phases of crystallographic space groups Pn3m and Ia3d, and a fluid isotropic phase, FI. The phase diagram of the beta-Xyl(Phyt)/water system is similar to that for the 1-monooleylglycerol (MO)/water system, suggesting that the phase behavior is largely determined by the overall molecular shape rather than the details of surfactant molecular structure. Moreover, the structural parameters of the beta-Xyl(Phyt) liquid crystals are also similar to those of the MO/water, due primarily to the similar molecular dimensions of two molecules. As compared to the MO/water system, however, the beta-Xyl(Phyt)/water system displays a lower value of TK ( approximately 8.(5) degrees C) and a wider temperature window for the mesophases (8.(5)-120 degrees C). Moreover, beta-Xyl(Phyt) is chemically more robust than MO, as the ether linkage is more stable against hydrolysis than the ester linkage and the phytanyl chain is fully saturated.     

Kinetics of lamellar-to-cubic and intercubic phase transitions of pure and cytochrome c containing monoolein dispersions monitored by time-resolved small-angle X-ray diffraction

We investigated the effect of incorporation of a small aqueous peripheral membrane protein (cyt c) into the three-dimensional periodic nanochannel structures formed by the lipid monoolein (MO) on its rich phase behavior as a function of temperature, pressure, and protein concentration using synchrotron X-ray small-angle diffraction. By simultaneous use of the pressure-jump relaxation technique and time-resolved synchrotron X-ray diffraction, we also studied the kinetics of various lipid mesophase transformations of the system for understanding the mechanistic pathways of their formation influenced by the protein-lipid interactions. Cyt c incorporated into the bicontinuous cubic phase Ia3d of MO has a significant effect on the lipid structure and the pressure stability of the system already at low protein concentrations. Concentrations higher than 0.2 wt % of cyt c led to an increase in interfacial curvature due to interaction of the protein with the lipid headgroups. This promotes the formation of a new, probably partially micellar cubic phase of crystallographic space group P4(3)32. Upon pressurization, the P4(3)32 phase undergoes a phase transition to a cubic Pn3m phase with smaller partial specific volume. Increase in protein concentration increases the pressure stability of the P4(3)32 phase. The formation of this phase from the cubic phase Pn3m is a slow process taking many seconds and having a time lag in the beginning. It seems to occur as a two-state process without ordered intermediate states. At temperatures above 60 degrees C, the P4(3)32 phase is unable to accommodate the unfolded protein and transforms to a bicontinuous cubic Ia3d phase. Time-resolved small-angle X-ray scattering studies show that the L(alpha) --> Ia3d transition in pure MO dispersions under limited hydration conditions occurs within a time interval of 1 s at 35 degrees C preceded by a lag phase of 1.5 s. The Ia3d cubic phase initially forms with a much larger lattice constant due to hydration and experiences an initially lower curvature that relaxes within about 1 s. Interestingly, no other cubic phases are involved as intermediates in the transition, i.e., the gyroid cubic phase is able to form directly from the L(alpha) phase. The mechanism behind the L(alpha) --> Ia3d transition in pure MO dispersions has been discussed within the framework of recent stalk models for membrane fusion. In the presence of cyt c, the L(alpha) --> Ia3d transition is much slower. The rather long relaxation times of the order of seconds are probably due to a kinetic trapping of the system and limitation by the transport and redistribution of water and lipid in the evolving new lipid phases. We also studied the transition from the pure lamellar L(alpha) phase to the Ia3d-P4(3)32 two phase region and observed a rather complex transition behavior with transient lamellar and cubic intermediate states.     

SAXS investigation of a cubic to a sponge (L3) phase transition in self-assembled lipid nanocarriers

The encapsulation and release of peptides, proteins, nucleic acids, and drugs in nanostructured lipid carriers depend on the type of the self-assembled liquid-crystalline organization and the structural dimensions of the aqueous and membraneous compartments, which can be tuned by the multicomponent composition of the systems. In this work, small-angle X-ray scattering (SAXS) investigation is performed on the 'melting' transition of the bicontinuous double diamond cubic phase, formed by pure glycerol monooleate (MO), upon progressive inclusion of varying fractions of pharmaceutical-grade glycerol monooleate (GO) in the hydrated system. The self-assembled MO/GO mixtures are found to form diamond (Pn3m) inverted cubic, inverted hexagonal (H(II)), and sponge (L(3)) phases at ambient temperature in excess of aqueous medium without heat treatment. Mixing of the inverted-cubic-phase-forming MO and the sponge-phase-forming GO components, in equivalent proportions (50/50 w/w), yields an inverted hexagonal (H(II)) phase nanostructured carrier. Scattering models are applied for fitting of the experimental SAXS patterns and identification of the structural changes in the aqueous and lipid bilayer subcompartments. The possibility of transforming, at ambient temperature (20 °C), the bicontinuous cubic nanostructures into inverted hexagonal (H(II)) or sponge (L(3)) mesophases may facilitate novel biomedical applications of the investigated liquid crystalline self-assemblies.     

On the microstructures of ethyl acetate/monoolein/water

A phase diagram, describing the behavior of the polar lipid monoolein (MO), water, and ethyl acetate (EtAc), is here presented as well as results from small angle X-ray scattering. MO is found to have a solubility of 60 wt.% in EtAc at 20 °C. No macroscopic aggregation of MO can, initially, be detected in the binary MO/EtAc solution even though MO forms solid crystals in concentrated samples when times goes by. In case of the ternary system small amounts of water, mainly bound to the lipid head groups, can be incorporated in the liquid EtAc/MO phase as water has a limited solubility in EtAc. For EtAc/water mass ratios below 2/3 EtAc is present into the reversed bicontinuous cubic and lamellar phases present in the binary MO/water system. To conclude, EtAc is mainly partitioned to the lipid membranes, with minor effects on spontaneous curvature. Hence, simple EtAc-addition has an effect similar to dehydration. For EtAc/water ratios above 2/3 the liquid crystalline phases dissolve. The phase behavior is here discussed in view of related phase behaviors for water-miscible solvent/MO/water systems. For instance, an interpretation of the swelling behavior of the sponge phase (L₃), present in the water-miscible solvent(s)/MO/water systems, shows that solvents partitioned to the polar domains strongly increases the spontaneous curvature of the MO-films. The reason is probably weaker hydrophobic interactions in interfacial regions. As expected, in case of water-miscible solvents, the ternary phase behaviors can be understood by consider water and water-miscible solvent as one “mixed solvent”.     

Nanostructural studies on monoelaidin-water systems at low temperatures

In recent years, lipid based nanostructures have increasingly been used as model membranes to study various complex biological processes. For better understanding of such phenomena, it is essential to gain as much information as possible for model lipid structures under physiological conditions. In this paper, we focus on one of such lipids--monoelaidin (ME)--for its polymorphic nanostructures under varying conditions of temperature and water content. In the recent contribution (Soft Matter, 2010, 6, 3191), we have reported the phase diagram of ME above 30 °C and compared with the phase behavior of other lipids including monoolein (MO), monovaccenin (MV), and monolinolein (ML). Remarkable phase behavior of ME, stabilizing three bicontinuous cubic phases, motivates its study at low temperatures. Current studies concentrate on the low-temperature (<30 °C) behavior of ME and subsequent reconstruction of its phase diagram over the entire temperature-water composition space (temperature, 0-76 °C; and water content, 0-70%). The polymorphs found for the monoelaidin-water system include three bicontinuous cubic phases, i.e., Ia3d, Pn3m, and Im3m, and lamellar phases which exhibit two crystalline (L(c1) and L(c0)), two gel (L(β) and L(β*)), and a fluid lamellar (L(α)) states. The fluid isotropic phase (L(2)) was observed only for lower hydrations (<20%), whereas hexagonal phase (H(2)) was not found under studied conditions. Nanostructural parameters of these phases as a function of temperature and water content are presented together with some molecular level calculations. This study might be crucial for perception of the lyotropic phase behavior as well as for designing nanostructural assemblies for potential applications.     

Lyotropic mesomorphism of rare-earth trisalkylsulphates in the water-ethylene glycol system

Lyotropic mesophases were observed for rare-earth trisdodecylsulphates, Ln(C₁₂H₂₅SO₄)₃ (Ln = Y, La, Ce-Lu, except Pm) in the presence of ethylene glycol or water (or mixtures thereof) as the solvent. In ethylene glycol, a normal hexagonal phase and a cubic phase could be detected, whereas in water a normal hexagonal phase and a lamellar phase were found. Thus by changing the solvent, it is possible to obtain different supramolecular organizations within a series of lanthanide-containing metallomesogens.     

Hydrostatic pressure effects on a hydrated lipid inverse micellar Fd3m cubic phase

Over a range of hydration, unsaturated diacylglycerol/phosphatidylcholine mixtures adopt an inverse micellar cubic phase, of crystallographic space group Fd3m. In this study hydrated DOPC:DOG mixtures with a molar ratio close to 1 : 2 were examined as a function of hydrostatic pressure, using synchrotron X-ray diffraction. The small-angle diffraction pattern at atmospheric pressure was used to calculate 2-D sections through the electron density map. Pressure initially has very little effect on the structure of the Fd3m cubic phase, in contrast to its effect on hydrated inverse bicontinuous cubic phases. At close to 2 kbar, a sharp transition occurs from the Fd3m phase to a pair of coexisting phases, an inverse hexagonal H(II) phase plus an (ordered) lamellar phase. Upon increasing the pressure to 3 kbar, a further sharp transition occurs from the H(II) phase to a (fluid) lamellar phase, in coexistence with the ordered lamellar phase. These transitions are fully reversible, but show hysteresis. Remarkably, the lattice parameter of the Fd3m phase is practically independent of pressure. These results show that these two lipids are miscible at low pressure, adopting a single lyotropic phase (Fd3m); they then become immiscible above a critical pressure, phase separating into DOPC-rich and DOG-rich phases.     

Effect of positively charged short peptides on stability of cubic phases of monoolein/dioleoylphosphatidic acid mixtures

To elucidate the stability and phase transition of cubic phases of biomembranes with infinite periodic minimal surface is indispensable from biological and physicochemical aspects. In this report, we investigated the effect of positively charged peptide-3K (LLKKK) and poly(L-lysine) on the phase stability of monoolein (MO) membranes containing negatively charged dioleoylphosphatidic acid (DOPA) (i.e., DOPA/MO membranes) using small-angle X-ray scattering. At first, the effect of peptide-3K on 10% DOPA/90% MO membrane in excess water, which is in the Q229 phase, was investigated. At 3.4 mM peptide-3K, a Q229 to Q230 phase transition occurred, and at >3.4 mM peptide-3K, the membrane was in the Q230 phase. Poly(L-lysine) (M(w) 1K-4K) also induced the Q230 phase, but peptide-2K (LLKK) could not induce it in the same membrane. We also investigated the effect of peptide-3K on the multilamellar vesicle (MLV) of 25% DOPA/75% MO membrane, which is in L(alpha) phase. In the absence of peptide, the spacing of MLV was very large (11.3 nm), but at > or = 8 mM peptide-3K, it greatly decreased to a constant value (5.2 nm), irrespective of the peptide concentration, indicating that peptide-3K and the membranes form an electrostatically stabilized aggregation with low water content. Poly(L-lysine) also decreased greatly the spacing of the 25% DOPA/75% MO MLV, indicating the formation of a similar aggregation. To compare the effects of peptide-3K and poly(L-lysine) with that of osmotic stress on stability of the cubic phase, we investigated the effect of poly(ethylene glycol) with molecular weight 7500 (PEG-6K) on the phase stability of 10% DOPA/90% MO membrane. With an increase in PEG-6K concentration, i.e., with an increase in osmotic stress, the most stable phase changed as follows; Q229 (Schwartz's P surface) --> Q224 (D) --> Q230 (G). On the basis of these results, we discuss the mechanism of the effects of the positively charged short peptides (peptide-3K) and poly(L-lysine) on the structure and phase stability of DOPA/MO membranes.     

pH-Dependent Release from Monoolein Cubic Phase Containing Hydrophobically Modified Chitosan

Monoolein (MO) cubic phase incorporating hydrophobically modified chosan (Hm chitosan) was prepared to obtain a pH-dependent release. Following calorimetric study, Hm chitosan had little effect on the crystal structure of MO cubic phase under acidic condition where Hm chitosan is readily soluble. At a higher pH (e.g., pH 9.0), however, the crystal structure of MO cubic phase was disturbed, possibly due to the insolubilization of Hm chitosan at the alkali condition. Whether the dye included in the cubic phase is anionic (amaranth) or cationic (methylene blue), the release from the cubic phase was suppressed as the pH of release medium increased. The structural change of cubic phase caused by the insolubilization of Hm chitosan, or the blockage of the water channel of the cubic phase by precipitated Hm chitosan would be responsible for the suppressed released.     

In vitro skin permeation of cubosomes containing water soluble extracts of Korean barberry

The monoolein (MO) cubic phases containing water soluble extract (WSE) from Berberis koreana (Korean barberry) were prepared by hydrating the molten MO with aqueous solutions of WSE (0.5, 1.0, and 1.5%). The phase transition temperature of cubic phase containing WSE (∼70°C) was almost the same as that of WSE-free MO cubic phase that indicates that WSE was immobilized in the water channels of the cubic phase and did not affect its structure. The release of WSE from the cubic phase fits the first order process. The cubosomes were obtained by micronizing the cubic phase in a sonicator using Pluronic F127 as a dispersant. The cubosomes were stable in size at the ethanol concentration ≲16%. When compared with WSE solution in phosphate-buffered saline (10 mM, pH 7.4), in vitro skin permeation of WSE in the cubosomes was enhanced by about two times.     

Lyotropic mesomorphism of rare-earth trisalkylsulphates in the water-ethylene glycol system

Lyotropic mesophases were observed for rare-earth trisdodecylsulphates, Ln(C₁₂H₂₅SO₄)₃ (Ln = Y, La, Ce-Lu, except Pm) in the presence of ethylene glycol or water (or mixtures thereof) as the solvent. In ethylene glycol, a normal hexagonal phase and a cubic phase could be detected, whereas in water a normal hexagonal phase and a lamellar phase were found. Thus by changing the solvent, it is possible to obtain different supramolecular organizations within a series of lanthanide-containing metallomesogens.     

On the microstructures of ethyl acetate/monoolein/water

A phase diagram, describing the behavior of the polar lipid monoolein (MO), water, and ethyl acetate (EtAc), is here presented as well as results from small angle X-ray scattering. MO is found to have a solubility of 60 wt.% in EtAc at 20 °C. No macroscopic aggregation of MO can, initially, be detected in the binary MO/EtAc solution even though MO forms solid crystals in concentrated samples when times goes by. In case of the ternary system small amounts of water, mainly bound to the lipid head groups, can be incorporated in the liquid EtAc/MO phase as water has a limited solubility in EtAc. For EtAc/water mass ratios below 2/3 EtAc is present into the reversed bicontinuous cubic and lamellar phases present in the binary MO/water system. To conclude, EtAc is mainly partitioned to the lipid membranes, with minor effects on spontaneous curvature. Hence, simple EtAc-addition has an effect similar to dehydration. For EtAc/water ratios above 2/3 the liquid crystalline phases dissolve. The phase behavior is here discussed in view of related phase behaviors for water-miscible solvent/MO/water systems. For instance, an interpretation of the swelling behavior of the sponge phase (L₃), present in the water-miscible solvent(s)/MO/water systems, shows that solvents partitioned to the polar domains strongly increases the spontaneous curvature of the MO-films. The reason is probably weaker hydrophobic interactions in interfacial regions. As expected, in case of water-miscible solvents, the ternary phase behaviors can be understood by consider water and water-miscible solvent as one “mixed solvent”.     

Formation of cubic phases from large unilamellar vesicles of dioleoylphosphatidylglycerol/monoolein membranes induced by low concentrations of Ca2+

We developed a new method for the transformation of large unilamellar vesicles (LUVs) into the cubic phase. We found that the addition of low concentrations of Ca(2+) to suspensions of multilamellar vesicles (MLVs) of membranes of monoolein (MO) and dioleoylphosphatidylglycerol (DOPG) mixtures (DOPG/MO) changed their L(alpha) phase to the cubic phases. For instance, the addition of 15-25 mM Ca(2+) to 30%-DOPG/70%-MO-MLVs induced the Q(229) phase, whereas the addition of > or =28 mM Ca(2+) induced the Q(224) phase. LUVs of DOPG/MO membranes containing > or =25 mol % DOPG were prepared easily. Low concentrations of Ca(2+) transformed these LUVs in excess buffer into the Q(224) or the Q(229) phase, depending on the Ca(2+) concentration. For example, 15 and 50 mM Ca(2+) induced the Q(224) and Q(229) phase in the 30%-DOPG/70%-MO-LUVs at 25 degrees C, respectively. This finding is the first demonstration of transformation of LUVs of lipid membranes into the cubic phase under excess water condition.     

Structural and rheological investigation of Fd3m inverse micellar cubic phases

In the present study we demonstrate that a bulk inverse micellar cubic phase of Fd3m structure can be obtained by adding a hydrophobic component, such as the food-grade limonene, to the binary system monolinolein/water in a well-defined composition. The Fd3m structure studied in this work had a very slow kinetics of formation, as a consequence of partitioning of water into two types of micelle populations with different sizes. The Fd3m structure formed at a ratio of limonene oil to total lipids of alpha = 0.4 is stable in the bulk up to a maximum hydration of 12.68 wt % water, beyond which it starts to coexist with dispersed water. At full hydration, by combining small-angle X-ray scattering and available topological models, the inverse micellar cubic phase of Fd3m structure was shown to be formed by 16 small micelles and 8 larger micelles per cubic lattice cell (Q227 group), with radii of the micellar polar cores ranging between 1 and 3 nm and 149-168 monolinolein molecules per micelle depending on the water content. The temperature dependence of the structural and rheological properties of the Fd3m mesophase was investigated using SAXS, rheology, and turbidimetry. It appeared that the Fd3m phase underwent crystallization below 18 degrees C and began melting in an inverse microemulsion (L2 phase) coexisting with water above 28.5 degrees C with complete melting obtained at 40-45 degrees C, as evidenced by SAXS and rheology. Macroscopic phase separation between the L2 phase and excess water was observed with time at higher temperatures. The investigation of the viscoelastic properties of the Fd3m inverse discrete micellar cubic phase revealed a rheological signature similar to that of the bicontinuous cubic phases Pn3m and Ia3d observed in homologous binary systems. However, the Fd3m phase presented a complex set of slower relaxation mechanisms leading to a shift by 1 order of magnitude of the dominant relaxation times and whole relaxation spectrum, as compared to those of inverse bicontinuous cubic phases. These findings have been tentatively explained by (i) the multiple relaxation of micelles upon deformation, (ii) the small hydration level of the Fd3m phase, and (iii) the low temperature at which this phase can be observed.     

甘油单油酸脂/水立方液晶体系流变性质的研究

Monoglyceride (MO) can form various liquid crystalline phases spontaneously in the presence of various amount of water at room temperature. The appropriate compositions from binary phase diagram of MO/H2O were selected to form cubic phases. The selected systems were studied at different salt concentrations and pH value using rheological methods. There was a weak effect of salt on viscoelastic properties of cubic phases formed from MO/H2O system. Hexagonal phase was formed when pH value was decreased or increased. The viscoelasticity of cubic phases was different from that of hexagonal liquid crystals. Rheological properties of MO/H2O cubic phases were stable at pH and salt concentration similar to physiological condition.     

Monoolein cubic phase containing alginate/cystamine gel for controlled release of epidermal growth factor

Monoolein (MO) cubic phase including alginate and cystamine in its water channel controlled the release of epidermal growth factor (EGF) by responding to changes in pH value and the reductive conditions of the release medium. The crosslinking degree of alginate gel with cystamine and the complex coacervation of alginate and EGF were investigated by using light scattering. TEM micrographs of cubic phases revealed MO bilayers along with water channels. Differential scanning calorimetry indicated that the cubic-to-hexagonal phase transition took place at 60.2?°C. Additives such as stearyl trimethyl ammonium chloride and cystamine decreased the transition temperature by a few to more than 10?°C. The release of EGF loaded in cubic phases was completed in 5?h and, thereafter, no significant additional release was observed. The release % of EGF loaded in MO cubic phase containing alginate and cystamine increased not only with the increase of pH but also glutathione concentration. The MO cubic phase containing alginate/cystamine gel can be used as a carrier for the delivery of peptide and protein drugs.     

Cubic phases and cubic phase dispersions in a phospholipid-based system

A cubic liquid crystalline phase forming system based on the phospholipid dioleoylphosphatidylethanolamine (DOPE) which is fortified with small amounts of PEGylated (poly(ethylene) glycol) glycerol monooleate (PEG(660)-GMO) is characterized. The cubic phase formed by the DOPE/PEG(660)-GMO/water system coexists with water in the dilute part of the phase diagram and can be fragmented into colloidal size particles with retained cubic phase structure.     

Monoolein cubic phase including in situ ionically gelled alginate and its salt-responsive release property

Salt-responsive monoolein (MO) cubic phase was prepared by in situ ionically gelling alginate contained in its water channels. On the TEM micrographs, bilayers, and water channels, characteristic of MO cubic phase were observed, and alginate and CaCl₂ had little effect on the structure. According to the differential scanning calorimetric thermogram, the cubic-to-hexagonal phase transition temperature of the cubic phase containing CaCl₂ solution was 46.8°C and it was much lower than that of the cubic phase containing distilled water, 60.5°C. The transition temperature was not significantly affected by alginate. The phase transition temperatures measured by the calorimetric analysis were in accordance with those determined by polarized optical microscopy. An initial burst release of dye (i.e., amaranth) was observed when the gelled alginate was not contained in the water channel of the cubic phase. A sustained release was obtained with the cubic phase containing the gelled alginate. The release of dye loaded in the cubic phase containing the gelled alginate was significantly promoted when the cubic phase came into contact with PBS (10?mM, pH 7.4), possibly because the multivalent cation (Ca²⁺) bound to alginate chains could be replaced by the monovalent cation (Na⁺).