Cubic and Sponge Phases in Ether Lipid–Solvent–Water Ternary

Article pubs.acs.org/Langmuir

Cubic and Sponge Phases in Ether Lipid−Solvent−Water Ternary Systems: Phase Behavior and NMR Characterization Hanne Evenbratt,†,∥ Lars Nordstierna,‡ Marica B. Ericson,§ and Sven Engström*,†,∥ †

Department of Chemical and Biological Engineering, Pharmaceutical Technology, Chalmers University of Technology, SE-41296 Gothenburg, Sweden ‡ Department of Chemical and Biological Engineering, Applied Surface Chemistry, Chalmers University of Technology, SE-41296 Gothenburg, Sweden § Department of Chemistry and Molecular Biology, University of Gothenburg, SE-40530 Gothenburg, Sweden S Supporting Information *

ABSTRACT: The phase behavior of 1-glyceryl monoleyl ether (GME) in mixtures of water and the solvents 1,5pentanediol (POL) or N-methyl-2-pyrrolidone (NMP) was investigated by ocular inspection, polarization microscopy, and small-angle X-ray diffraction (SAXD). Phase diagrams were constructed based on analyses of more than 200 samples prepared using the two different solvents at 20 °C. The inverse hexagonal phase formed by GME in excess of water was transformed into the cubic and sponge phase with the increasing amount of each solvent. Particularly POL allowed for the formation of an extended sponge phase area in the phase diagram, comprising up to 70% POL−water mixture. The phase behavior using NMP was found to be similar to the earlier investigated solvent propylene glycol. The extended sponge phase for the POL system was attributed to POLs strong surface/interfacial activity with the potential to stabilize the polar/apolar interface of the sponge phase. The cubic and sponge phases formed using POL were further studied by NMR in order to measure the partitioning of POL between the lipid and aqueous domains of the phases. The domain partition coefficient K (lipid domain/ aqueous domain) for POL in cubic and sponge phases was found to be 0.78 ± 0.14 and constant for the two phases.

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for crystallization of membrane proteins16 and is commercially available for that purpose. Not only can GMO form cubic phases in excess water, but also other polar lipids demonstrate similar phase diagrams, for example, phytantriol.19 However, the ether analogue of GMO, glyceryl monooleyl ether (GME), does not form a cubic phase spontaneously. Instead, an inverse hexagonal phase is obtained by combining GME with an excess of water, despite the similarity in the molecular structure of GMO and GME (see Figure 1).20 In a contribution by our group, we have demonstrated that PG added to GME in water seems to have the same effect on the inverse hexagonal phase as it has on the cubic phase in the GMO system. Consequently, the inverse hexagonal phase in the GME-water system is transformed to a sponge phase via a cubic phase in the presence of PG,21 which is interesting from a drug delivery perspective as the GME lipid is more stable than GMO. Recently, we have shown that topical drug delivery of the HCl salts of ALA and methyl-ALA in vivo (ALA = aminolevulinic acid, approved drug for photodynamic therapy of skin cancer) using cubic phases obtained from both GMO

INTRODUCTION One of the most remarkable self-assembly structures formed by polar lipids in aqueous media is the bicontinuous inverse cubic (liquid crystalline) phase, for example, the cubic phase obtained by combining monoolein (i.e., glyceryl monooleate, GMO) with excess water. Since the cubic phase based on GMO was first presented by Lutton in 1965,1 and its structure determined (for a comprehensive review, see ref 2), it has been used for various purposes such as drug delivery,3−5 biosensors,6,7 in drug partitioning studies,8−11 as template for solid nanoparticles,12 and for crystallization of membrane proteins.13 Also the so-called sponge phase can be used to crystallize membrane proteins, which is a liquid analogue to the cubic phase.14,15 The sponge phase may be obtained by adding certain water- and lipid-miscible solvents with slightly negative log Pow-values (P being the octanol−water partition coefficient) to the GMO−water system, such as propylene glycol (PG), dimethyl sulfoxide (DMSO), polyethylene glycol (PEG Mw ≈ 400−4000), and 2-methyl-2,4-pentanediol (MPD), where particularly the latter solvent has been used for membrane proteins crystallization13,16 (doctoral thesis, ref 17). The role of the solvent is most likely to affect the interfacial curvature of the GMO bilayer leading to an increased aqueous swelling and a subsequent phase transition from a cubic phase to a sponge phase.18 The GMO sponge phase has been successfully utilized © 2013 American Chemical Society

Received: July 18, 2013 Revised: September 21, 2013 Published: September 24, 2013 13058

dx.doi.org/10.1021/la402732a | Langmuir 2013, 29, 13058−13065

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Figure 1. Molecular structures of glyceryl monooleate or GMO (top), glyceryl monooleyl ether or GME (middle), propylene glycol or PG (bottom left), 1,5-pentanediol or POL (bottom middle), and N-methyl-2-pyrrolidone or NMP (bottom right).

and GME is superior to the commercial ointments.22 The clear advantage using the GME based system is because of the fact that the ether lipid is not subject to hydrolysis in the acidic environment provided by the ALA salts, which is the case for GMO. Combined with GME, the “bola” shaped skin penetration enhancer 1,5-pentanediol (POL)23,24 (Figure 1) showed the same improved delivery effect of ALA delivery as for PG.22 In the present work, we present detailed phase diagrams for two GME−solvent−water systems, the solvents being either POL (log Po/w −0.49) or NMP (log Po/w −0.46). The rationale for investigating POL is our current interest in topical drug delivery; while NMP is of interest, for example, for its applications as a polymer solvent for in situ forming implants.25 The large number of samples made for each system was analyzed by means of ocular inspection and between crossed polarizers. A limited number of samples were subject to polarization microscopy and small-angle X-ray diffraction (SAXD) to confirm the proposed structures. Some of the cubic and sponge phases formed with POL were further investigated by NMR spectroscopy. Such investigations were of interest, for example, because of the resemblance between POL and some of the solvents used in membrane protein crystallization (e.g., 1,4-butanediol26). The NMR study was undertaken in order to determine the “intrinsic” partitioning of POL between the lipid and aqueous domains, respectively, within the cubic and sponge phases.

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water were added using Eppendorf-pipettes (Eppendorf Research, Hamburg, Germany). Finally, the vials were sealed and the total weight measured. The samples were allowed to equilibrate at room temperature (approximately 20 °C). An additional sample was prepared with a start mixture of GME and an equimolar mixture of ethanol and 1-propanol. Water and GME were then stepwise added in accordance with the dotted lines shown in Figure 2. After reaching a sponge phase, a minuscule amount of crystal violet was added since this dye is known to adhere to lipid−water interfaces.16 Characterization of Phases. Ocular Inspection. Ocular inspections of the samples were performed regularly during a time period of several weeks. During the inspections, the number of phases present in each vial was determined at equilibrium, the relative amount of each phase in the vial was assessed, the order of the phases from top to bottom was noted, as well as rheological characteristics, and signs of birefringence in each phase. The inspection was performed at room temperature (20 °C) and atmospheric pressure. The lipid was assumed to behave as a single component during this study (which has been proven to be an accurate assumption in previous studies21). The analysis was guided by Gibb’s phase rule which, for a ternary system at room temperature reads f=3−p

(1)

where f is the number of degrees of freedom and p is the number of phases present at equilibrium. From this analysis, the one-, two-, and three-phase regions could be drawn in the phase diagrams. The uncertainty of the phase borders drawn is approximately 3% due to the coarse concentration mesh size. It turned out that some two- and three-phase regions were negligibly small at high lipid content (represented by at most one sample). These regions have been omitted from the results, since our main interest has been the solventand water-rich parts of the systems. SAXD. A limited number of samples were subject to SAXD analysis to determine the space group and the lattice parameter of the samples. The equipment used was a Kratky camera (Hecus X-ray systems, Graz, Austria), equipped with a position-sensitive, 1024-channel detector (Mbraun, Garching, Germany). Cu Kα radiation (λ = 0.1542 nm) was obtained from an X-ray generator (Philips, PW 1830/40, The Netherlands) operating at 50 kV and 40 mA. The analysis was performed at room temperature (20 °C). Vacuum was applied to minimize scattering from air. The distance from the sample to the detector was 275 mm and the run time typically 1800 s. The instrument was calibrated using crystalline tristearin showing a lamellar structure and a lattice parameter of 4.50 nm.

MATERIALS AND METHODS

Chemicals and Materials. GME 95% purity (Nikko Chemicals CO Ltd., Tokyo, Japan) was kindly provided as a gift from Jan Dekker International, France. NMP, ethanol, 1-propanol, and crystal violet were purchased from Sigma-Aldrich (Stockholm, Sweden). POL (98% purity) was provided by Acros Organics (Geel, Belgium). For the NMR study, D2O was obtained from Sigma-Aldrich (Stockholm, Sweden) and hexamethyldisilazane (≥98.0%) from Fluka (Stockholm, Sweden). Glass vials (0.7 mL) and vial-caps (NTK-kemi, Uppsala, Sweden) were applied for sample preparation. All water used was of Milli-Q grade, and the chemicals were all used as supplied. Preparation of Samples. More than 200 samples (approximately 500 mg each) were manually prepared for each system using a concentration “grid” of 5% (all percentages indicates wt %). First GME was melted and weighed into the vials. Thereafter POL or NMP and 13059


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NMR. All 1H NMR spectra were acquired using a Varian MR 400 spectrometer (Palo Alto, CA). Each spectrum was collected using a relaxation delay of 10 s and one 45° RF-pulse followed by 2.56 s acquisition time with 6410 Hz spectral width in the frequency domain. All NMR tubes contained an inserted and sealed capillary with pure hexamethyldisilazane together with the sample. The methyl signal of hexamethyldisilazane served as an external reference for the chemical shift value. Complementary 2H spectra were acquired for the inverse hexagonal, the inverse cubic, and the sponge phase where the water content had been replaced by an equivalent amount of D2O. These spectra were recorded using a Bruker Avance 600 spectrometer (Bruker, Solna, Sweden) . All NMR experiments in this work were carried out at 25 °C. For the spectral analysis and assignment of the chemical shifts, the software program MestReNova version 8.1.2 (Mestrelab Research, Santiago de Compostela, Spain) was used.

number (N > 200) of samples. The size of each one-, two-, and three-phase region could, however, only be determined within a few percent. In some cases, narrow two- and three-phase regions have been omitted from the phase diagrams because of limited number of samples in those regions. None of the samples demonstrated presence of more than three phases at equilibrium, which makes Gibbs’ phase rule a useful tool taking the purity of the lipid into consideration (GME 95% purity). The phases found were inverse hexagonal (H2), lamellar (Lα), inverse bicontinuous cubic (I2), sponge (L3) and liquid (L). All of these five one-phase areas were identified in the phase diagram using POL as solvent; three liquid-crystalline and two liquid phases. However, when using NMP as solvent at room temperature, the lamellar phase was absent, and only four one-phase areas were identified. The order of which the liquid crystalline phases are found in the phase diagrams is similar for the two solvents; however, the relative sizes of the one phase areas and the amount of solvent necessary to reach these equilibrium areas differ. The phase behavior of the systems will be discussed in more detail below. The Liquid Phase (L). Each of the two systems shows one boomerang-shaped congruent liquid phase region extending from the water corner, to the GME corner with varying amounts of solvent. However, under the bend of the liquid phase region, a large two-phase region is identified in each system (most evident in the POL diagram, Figure 2), where the liquid phase is split in two liquid phases, one rich and one poor in lipid. The presence of these two-phase regions of the two systems implies the existence of a critical point somewhere along the liquid phase border. The solubility of lipid was generally found to be low in solvent−water mixtures below 80% POL and 60% NMP. Instead the lipid proved to “precipitate” in the solvent−water mixtures forming liquid crystalline and sponge phases. Increasing the solvent concentration further increases the lipid solubility and finally leads to total miscibility. This occurs when the solvent/water molecular ratio becomes around or above one. The water solubility in solvent−lipid mixtures showed to be typically 10% in the NMP system but less than 5% in the POL system. The relatively low capacity of POL−GME mixtures to incorporate water in the liquid phase is most probably due to the “surface/interfacial activity” of the POL molecule, which should lead to strong preference for the polar part of the GME molecule.27 This will in turn lead to formation of more flat structures such as the bicontinuous cubic and sponge phases due to a strong flattening effect caused by the bulky “bola” shaped POL. The partitioning of POL in cubic and sponge phases was investigated further by means of NMR (see below). Sponge Phase (L3). The second liquid phase, found to be more centrally positioned in each phase diagram, was the sponge phase. This phase is characterized by a somewhat higher viscosity and density, compared to the less water containing liquid phase. Moreover, SAXD measurements of the sponge phase resulted in a broad Bragg peak which reflects the meso structure in the isotropic liquid (see Figure 3). A comparison of the NMP and POL systems reveals a much larger phase area and more swollen sponge phase for the latter. NMP behaves in this respect like PG.21 As mentioned above, we attribute the relatively large swelling of the POL sponge phase to POL’s strong tendency to accommodate in the polar/apolar region of the sponge phase. It has earlier also been found that POL has surfactant-like properties and decreases the surface tension of

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RESULTS AND DISCUSSION General Phase Behavior. The obtained phase diagrams of the ternary GME−POL−water and GME−NMP−water systems at room temperature are presented in Figure 2. The phase diagrams are based on ocular inspection of a large

Figure 2. Phase behavior of two ternary systems, GME−POL−water (top) and GME−NMP−water (bottom), at 20 °C. The diagrams are drawn in consideration of Gibbs’ phase rule. Some two- and threephase regions at high lipid content have been omitted due to uncertainties as a result of limited number of samples (see Supporting Information S1 for sample diagrams). Letters denote: liquid phase, L; lamellar phase, Lα; reversed hexagonal phase, H2; cubic phase, I2; and sponge phase, L3. The dashed arrows in the POL phase diagram indicates how water (a to b) and GME (b to c) have been added to a start mixture of GME and an equimolar mixture of ethanol and 1propanol at point a (see text). 13060


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Figure 3. X-ray diffractogram recorded at 20 °C showing a typical scattering curve for a sponge phase of the POL system. Sample 1 in Figure 2: GME/POL/water = 35%/35%/30%. The broad Bragg peak is centered around the scattering vector q = 2π/d = 0.5 nm−1 which implies average repeating distance d in the order of 12−13 nm.

Figure 5. X-ray diffractogram recorded at 20 °C showing a diffraction pattern for a sample in the cubic phase region of the NMP system. Sample 3 in Figure 2: GME/NMP/water = 65%/13%/22%. The Bragg reflections have been indexed to two cubic phases with space group Pn3m (triangles, lattice parameter 9.0 nm) and space group Ia3d (squares, lattice parameter 14.3 nm).

water from 73 to 45−50 mN m−1 at and above 20% POL in water.27 The swelling capacity of the GME/POL/water sponge phase may be of interest for use in, for example, membrane protein crystallization. The commercially available lipidic sponge phase (LSP) screen, based on GMO, contains typically 30% water.16 A screen based on GME and POL (or rather 1,4-butanediol used in the crystallization of a G-protein coupled receptor26) could allow for screen solutions of both lower and higher pH than for GMO based screens which can only be used in the pH range 5.5−9 due to the risk for ester hydrolysis.16 Another advantage with the GME (and GMO) sponge phases is their nonionic nature, which make them less sensitive to salt. They are also stable over a large temperature range (see below). Cubic Phase (I2). At NMP concentrations higher than 10% and POL at least 5%, even a small amount of lipid in excess water will form the highly viscous, isotropic (inverse bicontinuous) cubic phase. This cubic phase exists to a NMP and POL content of about 35% and 25%, respectively. SAXD results are shown in Figures 4 and 5. Cubic phases of space group Pn3m were the most frequently found in these samples as shown for the POL containing sample in Figure 4. However,

SAXD data also imply the existence of another space group, Ia3d, which is consistent with the existence of two cubic phases present in the GMO−water system.28 Both space groups are shown in Figure 5 for the NMP-containing cubic sample. The shift between Pn3m and Ia3d can be extremely sensitive because of the small cubic one phase areas. The high viscosity and adhesive properties of the cubic liquid crystalline phase makes it appealing for topical, and in particular dermal, drug delivery. The gel-like texture makes it easy to apply to the skin surface. Recently published results show enhanced dermal drug delivery when applying the GME− POL−water system for use in photodynamic therapy.22 Lamellar Phase (Lα). The lamellar phase is a mucouslike, birefringent, liquid crystalline phase which has an anomalous position in the GME and GMO systems.29 In this study, the lamellar phase was only found for the POL system (Figure 6),

Figure 6. X-ray diffractogram recorded at 20 °C showing the diffraction pattern for a sample in the lamellar region of the POL system. Also, two inset images of the lamellar phase obtained in the polarizing microscope (magnification 50×). Sample 4 in Figure 2: GME/POL/water = 78%/9%/13%.

and situated between the cubic phase and the lipid rich liquid phase of the phase diagram; see Figure 2. The lamellar phase of GME (and GMO) typically shows only one Bragg peak in Xray diffractograms, as depicted in Figure 6. Inset in the figure is also two images of the lamellar phase obtained in a polarizing microscope. To the left, the black and white lamellar structure

Figure 4. X-ray diffractogram recorded at 20 °C showing a diffraction pattern for a sample in the cubic phase region of the POL system. Sample 2 in Figure 2: GME/POL/water = 57%/9%/34%. The Bragg reflections have been indexed to one cubic phase with space group Pn3m (triangles, lattice parameter 9.7 nm). 13061


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is shown with malteser crosses starting to form. The lamellar phase is very temperature sensitive which was evident when the sample started to phase separate because of the increased temperature caused by the light source (right image in Figure 6). The existence of a lamellar phase in the POL system may be of interest for the use of the system for membrane protein crystallization. It turns out that most membrane proteins crystallized by means of the lipidic cubic phase or lipidic sponge phase techniques so far have been of the so-called lamellar type I. This implies that the lamellar phase may play a vital role in the crystallization process.30 Looking at the lipid corner from the water-solvent edge of the POL phase triangle reveals that, “behind” the H2, I2, and L3 phases, the Lα phase is found at lower solvent/water, and consequently higher lipid, content. In the original crystallization setup by Landau and Rosenbusch for bacteriorhodopsin it was obvious that the initial cubic phase lost its water due to the osmotic effect caused by the added screen solution,13,15 thus surrounding the crystals with a lamellar layer. Inverse Hexagonal Phase (H2). GME forms an inverse hexagonal phase with approximately 25% water and the hexagonal structure can only accommodate a few percent of the solvents as revealed by the phase diagrams in Figure 2 (see Supporting Information S2 for SAXD pattern and an image obtained in the polarizing microscope). The transformation to bicontinuous cubic phases at increased solvent contents is a strong indication of the flattening effect of the solvents at the polar/apolar interface. Although the hexagonal phase has not been in focus of the present study, it should be noted that by adding (e.g., spraying, dispersing, etc.) cubic or sponge phases to excess water will transform these phases to an inverse hexagonal phase (hexosomes31) according to the phase diagrams. Temperature Dependence. The temperature stability of the H2, I2, and L3 phases in two- or three-phase regions along the solvent/water composition line at 20% GME was studied in the temperature range 18−50 °C (see Figure 7). As shown by the figure, the phases are relatively insensitive to temperature changes in the range from room to body temperature. It was found that the liquid phase area increases at higher temperatures for both systems. Also the sponge phase area increases mainly at the expense of the cubic phase. For both systems a single one phase area was found when heating the samples with the highest amount of solvent to 50 °C, indicated by the black square in Figure 7. All other samples contained two or three phases. In the POL containing system the sponge phase dominates the temperature diagram, though it is evident that any phase formed is stable within the temperature range. When lowering the temperature a lamellar phase was found at high POL concentration. This is not surprising considering POL’s affinity for the polar/apolar interface. In the NMP containing temperature depending phase diagram more three phase areas were observed. This agrees with the relatively small one phase areas found in the phase diagram. At critical solvent concentrations and temperatures phase changes occur. For all samples, the phase distribution found at room temperature reoccurred after the temperature alteration. The temperature robustness of these systems is of importance for, for example, pharmaceutical applications. NMR Studies of POL Phases. When comparing the GME− solvent−water phase diagrams based on POL or NMP it is

Figure 7. The left part of the phase diagrams shows phase temperature dependence. A water rich liquid phase was present in all samples, with one or two other above. The latter are shown in the diagram. The temperature was varied between 18 and 50 °C. The GME, solvent, water phase diagrams, first shown in Figure 2, are here marked with the concentrations used to study the effect of temperature alteration on phase behavior. Letters, symbols (and grayscale) denote: liquid phase, L (○); lamellar phase, Lα (×); reversed hexagonal phase, H2 (△); cubic phase, I2 (□); and sponge phase, L3 (△). The black corner denotes one single liquid phase.

evident that POL behaves differently in the systems compared to NMP, which was mentioned above. As POL may be of interest in, for example, protein crystallization and dermal drug delivery, a more detailed investigation of its distribution in the phases was of interest and NMR was undertaken. The 2H NMR signal patterns of the inverse hexagonal, the inverse cubic, and the sponge phase respectively were recorded to confirm the microstructural anisotropy and isotropy of these phases.32 The anisotropic symmetry of the inverse hexagonal phase was reflected by a quadrupolar splitting of the D2O signal, whereas the inverse cubic and sponge phases provide narrow 2H singlets due to absence of phase anisotropy. The result was in concurrence with previously published phase behavior.33 Due to severe signal broadening, the 1H spectrum of the inverse hexagonal phase was not studied in this work. For the inverse cubic and in the sponge phase, single 1H resonances from the α, β, and γ hydrogens respectively of POL (see Figure 1) showed that any exchange of this alcohol between aqueous and lipid domains occur on a time-scale faster than ten milliseconds. This time correlate to the frequency difference between NMR signals of POL in these domains. The observed 13062


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1

At this stage in our work, we are only interested in the chemical shift values of POL, in H2O and GME, as a function of weight fraction. It was thus motivated to simply adapt a global curve fitting on the experimental data of each specific proton using two second-degree polynomials.

H NMR signal can thus be regarded as a population average of POL in the aqueous and lipid domains and expressed by obs obs aq lip lip δ POL = pPOL δ POL + pPOL δ POL

paq POL

(2)

plip POL

where and are the fractions of the total amount POL distributed in the aqueous and lipid domains, respectively, with lip paq POL + pPOL = 1. Let us consider a system with weight fraction H2O, GME, and POL represented by xH2O, xGME, and xPOL. Water and GME are considered to be immiscible. The total weight fraction xPOL of POL in the sample is expressed by the phase equilibrium as aq lip x POL = x POL + x POL

aq POL 2⎫ ⎧ ⎪ δ POL = δ POL + a(x − 1) + b(x − 1) ⎪ ⎬ ⎨ ⎪ lip POL 2⎪ ⎩ δ POL = δ POL + c(x − 1) + d(x − 1) ⎭

Note that x in eq 7 expresses the weight fraction POL in each specific binary system. Using the calculated coefficients a−d, we can describe the chemical shift of POL in water and GME, from infinite solvent environment to pure POL and the fitted curves are shown in Figure 8. By observing the chemical shifts of POL in the inverse cubic and sponge phases, one can calculate its weight fraction xaq POL (including the entire system) in the aqueous domain by using eqs 2−4 and 7. The parameter x in eq 7 is here replaced according to

(3)

and we can write aq pPOL =

aq x POL x POL

(4)

We may here conclude that a prior knowledge about the chemical shift in each environment would provide the equilibrium fraction of POL in each of the two domains. The chemical shift in each environment is however influenced by the internal content of POL as given by the two following equations. aq POL H 2O δ POL = ϕ0,POLδ POL + ϕ0,H Oδ POL

(5)

lip POL GME δ POL = ϕ0,POLδ POL + ϕ0,GMEδ POL

(6)

2

(7)

⎧ ⎫ x aq ⎪ ⎪ x (aqueous) ≡ aq POL x POL + x H2O ⎪ ⎪ ⎬ ⎨ aq x POL − x POL ⎪ ⎪ ⎪ ⎪ x (lipid) ≡ x aq POL − x POL + x H 2O ⎭ ⎩

(8)

The equilibrium partitions of POL in the aqueous and lipid domains are now given and can be calculated by a population averaged chemical shift. The lipid phase evolves from inverse cubic geometry to sponge upon addition of POL. Most probably this is due to the intrusion of the alcohol between the GME molecules at the polar/apolar interface, and this effect provides less curvature of the GME head groups toward the water domain. By chemical shift studies we have determined the domain partition coefficient of POL between the GME and water domains.

where ϕ0,i is the molar volume fraction of substance i and the equations holds only for complete mixing of POL with H2O and GME. The density of POL, H2O, and GME are similar and one could therefore believe that the chemical shift of POL in H2O or GME is linearly varied by the weight fraction. This is clearly not the case for any of the protons as seen in Figure 8.

K=

lip lip lip c POL x POL /x POL + xGME = aq aq aq c POL x POL /x POL + x H 2O

(9)

The average K-value for the obtained data, using eq 9, resulted in K = 0.78 ± 0.14, thus indicating a slight preference for POL in water domains. The results are given in Figure 9 where the K-values for POL in cubic and sponge phases are plotted against weight percent POL in the samples. Each point

Figure 8. Chemical shift of the α (solid red square), β (solid green circle), and γ (solid blue up triangle) 1H NMR signals of POL in H2O (full symbols) and GME (open symbols). All signals have been calibrated to external reference. The full and dashed lines correspond to second-degree polynomial curve fitting as explained in the text.

Here we see a curved trend of chemical shifts with increased content of POL, notably more for H2O than for GME. Most probably, and also discussed earlier,34 the reason for curvature is nonideal mixing of the binary solution. Volume excess of POL in water has, to our knowledge, not been presented in the literature; however, other studies have confirmed nonideality of mixing the two solvents.35,36

Figure 9. Experimentally determined partition coefficient (K) for POL between lipid and water domains within the cubic and sponge phases over the POL phase concentration. 13063


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evident comparing the effect of POL with its simple alcohol analogues, ethanol or 1-propanol, where sponge phases are less extended. We believe that the present study is of great value for the understanding of mechanisms behind membrane protein crystallization processes using cubic or sponge phases by means of screen agents such as 1,4-butanediol.

represents the average and standard deviation of data collection from α, β, and γ protons. It is apparent from the linearity in Figure 9 that the partition of POL between the two domains is constant likewise to the case for two macroscopic phases. This finding holds regardless of the position in the ternary phase diagram and regardless of the phase in the diagram. This is to our knowledge the first time the partition of solvent in the cubic and sponge phases are revealed in this way. It may provide valuable clues to understand the mechanism behind, for example, protein crystallization using screening agents similar to POL, such as 1,4-butanediol.15 Replacing POL with Ethanol and 1-Propanol. In an attempt to elucidate the preference of POL for the polar region of GME, we made a series of samples where POL was replaced with an equimolar mixture of ethanol and 1-propanol as a way to show the effect of a split POL molecule. The equimolar mixture was mixed with GME (1:1 by weight) and water added in several steps (see Figure 2, dashed arrows indicate addition of water and GME). It turned out that at least 38.4% water could be added with the liquid phase (L) preserved which should be compared with less than 5% for POL. In the range of 41.0−59.1% water, two liquid phases were present where the relative volume of the denser phase increased upon water addition. We interpret this two-phase region as consisting of a lipid rich top and a lipid poor bottom phase. At 63.7% water, three liquid phases were found in equilibrium, the higher density bottom liquid having the same volume as the upper two liquids together. We interpret the middle liquid phase as a sponge phase since it grows at the expense of the top phase upon further addition of water. At 66.3% water, the top phase had disappeared and two liquids were at equilibrium (point b in the POL diagram in Figure 2). We then started to add GME, and at 52.4% water (point c in Figure 2) the top (sponge) liquid phase occupied about 80% of the volume of the sample. Finally, we added a dye, crystal violet, to the sample which is known to adhere strongly to polar− apolar interfaces and could see that the dye partitioned into the upper phase almost exclusively. We take this observation as a very strong indication of a sponge phase.16 Taken together these results indicate that the mixture of ethanol and 1-propanol seem to have a very strong flattening effect on the polar/apolar interface in GME−water mixtures which is revealed by a sponge phase around 10% alcohol content. At an alcohol concentration of about 30% the system has converted to a liquid phase, L according to the present notation, where the molecules are more or less randomly mixed. This is in sharp contrast to POL which obviously stabilizes the polar/apolar interface in the system. This is revealed by a sponge phase extending to high POL contents.

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ASSOCIATED CONTENT

S Supporting Information *

S1: Sample diagrams. The symbols denote liquid (○), lamellar (+), reverse hexagonal (△), cubic (□) and sponge (◊) phases. S2: X-ray diffractogram recorded at 20 °C showing the diffraction pattern for a sample in the hexagonal region. Also, an inset image of the hexagonal phase obtained in the polarizing microscope (magnification 50×). GME/water = 75%/25%. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +46 31 772 2765; Fax: +46 31 772 3418; E-mail address: [email protected]. Author Contributions ∥

H.E. and S.E. contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge Peter Larsen and Christa Nilsson for technical assistance and the Swedish Research Council, VR for funding (ref no. 621-2008-4803). NMR experiments have been carried out at the Swedish NMR Centre. The project was performed within Centre for Skin Research, SkinResQU.

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ABBREVIATIONS GME, 1-glyceryl monoleyl ether; GMO, glyceryl monooleate; POL, 1,5-pentanediol; NMP, N-methyl-2-pyrrolidone; PG, propylene glycol; DMSO, dimethyl sulfoxide; PEG, polyethylene glycol; MPD, 2-methyl-2,4-pentanediol; SAXD, smallangle X-ray diffraction; ALA, aminolevulinic acid; PDT, photodynamic therapy; NMR, nuclear magnetic resonance

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REFERENCES

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CONCLUSIONS Ternary systems comprising GME, water, and a solvent, that is, POL or NMP, show a rich variety of phases at room temperature. Of particular interest for biotechnical applications (e.g., drug delivery and membrane protein crystallization) are the phases formed in excess solvent−water mixtures, which goes from inverse hexagonal to inverse cubic and sponge phase upon increasing solvent content. While the NMP containing system shows a phase behavior similar to that of PG in a corresponding study, POL instead demonstrates an extended sponge phase up to high solvent levels. This is particularly 13064


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Cubic and Sponge Phases in Ether Lipid–Solvent–Water Ternary

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