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Langmuir 1997, 13, 5476-5483
Structural Effects, Mobility, and Redox Behavior of Vitamin K1 Hosted in the Monoolein/Water Liquid Crystalline Phases Francesca Caboi,† Tommy Nylander,*,‡ Valdemaras Razumas,§ Zita Talaikyte´,§ Maura Monduzzi,† and Ka˚re Larsson| Dipartimento di Scienze Chimiche, Universita` di Cagliari, Via Ospedale 72, 09124 Cagliari, Italy, Physical Chemistry 1, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, S-221 00 Lund, Sweden, Institute of Biochemistry, Mokslininku 12, 2600 Vilnius, Lithuania, and Camurus AB, Ideon Research Park, S-223 70 Lund, Sweden Received February 27, 1997. In Final Form: June 30, 1997X
The solubilization of vitamin K1 (VK1), a highly hydrophobic molecule, into a 1-monoolein/water (MO/W) system, is investigated by NMR self-diffusion, small angle X-ray scattering (SAXS), optical microscopy, and electrochemical methods. The various MO/W phases, namely L2, LR, CG, and CD, can accommodate different amounts of VK1. In particular, the LR and the cubic CG phases can solubilize up to 8 and 5 wt % VK1, respectively, without modifing the microstructure substantially. By contrast, the cubic CD phase can accommodate only about 1 wt % VK1. Larger addition of VK1 produces a transition from the lamellar and cubic phases to a reverse hexagonal phase HII, which in the MO/W binary system occurs only for the cubic phases and at temperatures above 80 °C. In practice the solubilization of VK1 induces almost the same phase transitions as would a temperature increase in the binary system. The SAXS and NMR self-diffusion data strongly suggest that the VK1 molecules are well intermingled with the MO hydrophobic chains. Consequently the swelling of the CG and LR phases does not seem to be affected by the amount of VK1 present. However, if a sufficient number of VK1 molecules has penetrated into the lipid bilayer, the local change of the bilayer curvature is so large that a transition from cubic or LR phase to a reverse hexagonal phase will occur. Electrochemical measurements indicate that, when solubilized in the cubic phase, the naphthoquinone group of VK1 reaches the bilayer/aqueous interface during the redox cycle, as the formal redox potential of the group is pH-dependent. The potential use of MO/W cubic phases with electrochemically active bilayer components in bioanalytical systems is discussed.
Introduction Polar lipid/aqueous systems are widely used as model matrices to mimic biological processes where the phase behavior of lipids plays a mediating role. In various pharmaceutical, biotechnical, and food applications, the liquid crystalline phases formed by polar lipids in an aqueous medium, represent useful host systems for drugs, enzymes, vitamins, or any active molecule.1 The hydrophilic/hydrophobic nature of the guest molecule determines whether the molecule will be preferentially located in the polar aqueous domain or in the apolar hydrocarbon domain. The structure of lyotropic liquid crystals in lipid/water systems has been thoroughly characterized by a variety of techniques, mainly small angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR), and electron and optical microscopy. The occurrence of isotropic solutions that contain micelles or vesicles, as well as anisotropic liquid crystalline phases like lamellar phases, hexagonal phases, and even transparent isotropic phases with a cubic three-dimensional lattice has been well documented in many lipid/water systems.1-6 The formation of a par* Author to whom correspondence should be addressed. E-mail:
[email protected]. † Universita ` di Cagliari. ‡ Lund University. § Institute of Biochemistry. | Camurus AB. X Abstract published in Advance ACS Abstracts, August 15, 1997. (1) Larsson, K. LipidssMolecular Organization, Physical Functions and Technical Applications; The Oily Press Ltd.: Dundee, 1994.
S0743-7463(97)00218-7 CCC: $14.00
ticular phase can be rationalized on the basis of the geometrical packing properties of the lipid in the particular environment.7 These properties can be easily expressed by the packing parameter, ν/al, where ν is the volume of the hydrophobic chain, a is the head group area, and l is the chain length.7,8 It is important to stress that the packing parameter is dependent not only on the structure of the amphiphile but also on particular conditions like ionic strength, temperature, and, as we will illustrate in this study, the interaction with other molecules accommodated in the lipid structure. Lately cubic structures have received increased attention; in particular, their microstructures and their significance in biological systems have been investigated.2-4,6,9 Several biological membrane lipids form cubic phases, and cubic phases participate in various biological processes. For example, it has been suggested that acylglycerols assume a cubic structure in the intermediate state of fat digestion.2,10,11 Recent studies demonstrated (2) Mariani, P.; Luzzati, V.; Delacroix, H. J. Mol. Biol. 1988, 204, 165. (3) Lindblom, G.; Rilfors, L. Biochim. Biophys. Acta 1989, 988, 221. (4) Larsson, K. J. Phys. Chem. 1989, 93, 7304. (5) Fontell, K. Adv. Colloid Interface Sci. 1992, 41, 127. (6) Seddon, J. M. Biochim. Biophys. Acta 1990, 1031, 1. (7) Mitchell, D. J.; Ninham, B. W. J. Chem. Soc., Faraday Trans. 2 1981, 77, 601. (8) Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. J. Chem. Soc., Faraday Trans. 2 1976, 72, 1525. (9) Hyde, S.; Andersson, S.; Larsson, K.; Blum, Z.; Landh, T.; Lidin, S.; Ninham, B. W. The language of shape. The role of curvature in condensed matter: Physics, chemistry and biology; Elsevier: Amsterdam, 1997. (10) Patton, J. S.; Carey, M. C. Science 1979, 204, 145. (11) Patton, J. S.; Vetter, R. D.; Hamosh, B.; Borgstrøm, B.; Lindstrøm, M.; Carey, M. C. Food Microstruct. 1985, 4, 29.
© 1997 American Chemical Society
Vitamin K1 Hosted in the Monoolein-Water Phases
Figure 1. Molecular structures of vitamin K1 (VK1) and monoolein (MO).
that cubic phases can accommodate a wide range of globular proteins.2,12-18 In this context it is worth mentioning that cubic liquid crystalline phases with entrapped enzymes can be used to produce electrochemical biosensors.16,18 In addition cubic phases can be used in pharmaceutical applications for controlled drug release.1 Different types of cubic structures formed in aqueous systems have been identified.3-6,19 They depend on the particular type of lipid. Only bicontinuous cubic phases, comprised of curved nonintersecting bilayers organized to form two disjoint continuous water channels, will be considered here.4,20 If a plane is placed in the gap between the methyl end groups of the lipid bilayer of the cubic phase, the surface obtained can be described by an infinite periodic minimal surface (IPMS).4,21 The curvature of any surface is given by the two principal radii of curvature, R1 and R2. The average surface curvature 1/2(1/R1 + 1/R2) at any point of a minimal surface is zero by definition. The packing parameter, ν/al, for a lipid in such a curved bilayer is close to or slightly larger than unity and can be connected to the Gaussian curvature (1/R1R2) of the IPMS.22 Three types of IPMS’s, describing different cubic space groups, are important in lipid systems:4,21 the diamond (D) type (primitive lattice (Pn3m)), the gyroid (G) type (body-centered lattice (Ia3d)), and the primitive (P) type (body-centered lattice (Im3m)). We will focus here on the solubilization of vitamin K1 (VK1, Figure 1), a highly hydrophobic molecule, into the 1-monoolein (monooleoylglycerol)/water (MO/W) system, using NMR self-diffusion, SAXS, optical microscopy, and electrochemical methods. The phase behavior of the MO/W binary system has been thoroughly investigated.20,23-27 Reverse micellar phases, lamellar phases, (12) Ericsson, B.; Larsson, K.; Fontell, K. Biochim. Biophys. Acta 1983, 729, 23. (13) Buchheim, W.; Larsson, K. J. Colloid Interface Sci. 1987, 117, 582. (14) Portmann, M.; Landau, E. M.; Luisi, P. L. J. Phys. Chem 1991, 95, 8437. (15) Landau, E. M.; Luisi, P. L. J. Am. Chem. Soc. 1993, 115, 2102. (16) Razumas, V.; Kanapieniene´, J.; Nylander, T.; Engstro¨m, S.; Larsson, K. Anal. Chim. Acta 1994, 289, 155. (17) Razumas, V.; Larsson, K.; Miezes, Y.; Nylander, T. J. Phys. Chem. 1996, 100, 11766. (18) Nylander, T.; Mattisson, C.; Razumas, V.; Miezes, Y.; Ha˚kansson, B. Colloids Surf. A: Physicochem. Eng. Aspects 1996, 114, 311. (19) Luzzati, V.; Tardieu, A.; Gulik-Krzywicki, T.; Rivas, E.; ReissHusson, F. Nature 1968, 220, 485. (20) Lindblom, G.; Larsson, K.; Johansson, L.; Fontell, K.; Forse´n, S. J. Am. Chem. Soc. 1979, 101, 5465. (21) Andersson, S.; Hyde, S. T.; Larsson, K.; Lidin, S. Chem. Rev. 1988, 88, 221. (22) Hyde, S. T. J. Phys. Chem. 1989, 93, 1458. (23) Larsson, K. Nature 1983, 304, 664. (24) Hyde, S. T.; Andersson, S.; Ericsson, B.; Larsson, K. Z. Kristallogr. 1984, 168, 213. (25) Chung, H.; Caffrey, M. Biophys. J. 1994, 66, 377. (26) Landh, T. J. Phys. Chem. 1994, 98, 8453. (27) Briggs, J.; Chung, H.; Caffrey, M. J. Phys. II 1996, 6, 723.
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and two cubic phases, the diamond and the gyroid type, form the best characterized liquid crystalline phases that have been identified at room temperature in this system. A reverse hexagonal phase occurs above 80 °C. As an example of solubilization of a hydrophobic species, Moaddel et al. reported on vitamin E trapped in the lamellar phase of lecithin.28 This vitamin, which has a structure somewhat similar to that of VK1, can be solubilized into the lamellar structure, without any significant change of the interlayer spacings, up to a maximum content of 20% by weight. The ability of the lipid-soluble VK1 to participate in mitochondrial oxidative phosphorylation is well-known. In addition it may act as a physiological cofactor of cyclic electron transport in photosynthetic processes. The biological activity of VK1 is due to its ability to develop a reversible redox conversion, clearly a matter of considerable interest. Surprisingly, however, compared with the extensive electrochemical studies of related compounds (e.g., ubiquinones29), reports on VK1 are sparce. Of the few published results, electrochemical investigations of VK1 adsorbed onto the surface of pyrolytic graphite30 and incorporated into n-alkanethiol monolayers on a gold electrode31 can be mentioned. One reason for the paucity of studies on this system is that, up to now, there has not been a reliable membrane model available to monitor the redox properties of the vitamin. In this work that difficulty is removed. Electrochemical conversions of VK1 entrapped in the cubic liquid crystalline phase were studied by cyclic voltammetry. Experimental Section Materials. Monoolein (1-monooleoylglycerol, batch TS-ED 173), MO, containing 98.1% monoglycerides, was kindly provided by Danissco Ingredients, Brabrand, Denmark. Vitamin K1 (2methyl-3-phytyl-1,4-naphthoquinone) was purchased from Sigma and used as received. The water was distilled and passed through a Milli-Q water purification system (Millipore). Samples were prepared by weighing appropriate amounts of MO into glass vials, melting it at 40 °C, dissolving VK1 in the melted lipid, and then adding water. The lipid samples, which were kept in tightly sealed vials, were centrifuged several times at 40 and 25 °C before storing them at 25 °C for at least 3 weeks or until the samples were homogeneous. Measurements were performed once equilibrium was apparent. After 5 months small changes were observed upon visual inspection for some samples close to the phase boundaries; that is, for instance, three of the lamellar samples very close to the border with the cubic region became pure cubic or cubic and lamellar. Methods. The homogeneous liquid crystalline phases were observed by optical microscopy (Zeiss Axioplan II) in polarized light, at 25 °C, and compared with the typical textures of other surfactants.32,33 Self-diffusion experiments were performed on a Varian Unity 400 spectrometer, equipped with a superconducting 360 MHz Oxford wide bore magnet and a home-built pulsed magnetic field unit. The FT-PGSE technique, developed by Stejskal and Tanner,34 was used, and the experiments were carried out by varying the gradient pulse length (δ), while keeping the gradient strength (G) and the pulse intervals (∆) constant. (28) Moaddel, T.; Friberg, S. E.; Brin, A. Colloid Polym. Sci. 1996, 274, 153. (29) Moncelli, M. R.; Becucci, L.; Nelson, A.; Guidelli, R. Biophys. J. 1996, 2716. (30) Ksenzhek, O. S.; Petrova, S. A.; Kolodyazhny, M. V.; Oleinik, S. V. Bioelectrochem. Bioenerg. 1977, 4, 335. (31) Takehara, K.; Takemura, H.; Ide, Y. J. Electroanal. Chem. 1991, 308, 345. (32) Mandell, L.; Fontell, K.; Ekwall, P. In Ordered Fluids and Liquid Crystals; Porter, R. S., Johnson, J. F., Eds.; American Chemical Society: Washington, DC, 1967; p 891. (33) Rosevear, F. B. J. Am. Oil Chem. Soc. 1954, 31, 628. (34) Stejskal, E. O.; Tanner, J. E. J. Chem. Phys. 1965, 42, 288.
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The echo intensity decay as a function of δ is given by
I(δ) ) I(0) exp[-(γGδ)2D(∆ - δ/3)]
(1)
where D is the self-diffusion coefficient, I(0) is the echo intensity in the absence of the gradient, and γ is the gyromagnetic ratio. Self-diffusion coefficients were calculated by means of a twoparameter nonlinear fit of eq 1 to 15-20 different δ values. The error in the measurements is estimated to be smaller than (8%. X-ray diffractograms of the cubic and lamellar phases were obtained at 25 °C by means of two types of devices using Cu KR nickel-filtered radiation of wavelength 1.542 Å, a Guinier camera, in Luzzati setup,35 and a Kratky compact small angle system. The sample-to-film distance was 19.4 cm in the Luzzati camera, and the sample holder was as for Hernqvist.36 A film exposure time of 24 h was used. The Kratky system was equipped with a position sensitive detector (OED 50M from Mbraun, Graz, Austria) containing 1024 channels of width 51.3 µm. The radiation was provided by a Seifert IF 300 X-ray generator operating at 50 kV and 40 mA, and the sample-to-detector distance was 27.7 cm. Temperature control within 0.1 °C was achieved by using a Peltier element. Cyclic voltammograms were recorded by a PA-2 polarograph (Laboratorni pristroje, Czechoslovakia) in a three-electrode circuit using a 20 cm3 glass cell thermostated at 25 ((0.1) °C. Measurements were performed with a working electrode from a gold disk (0.194 cm2) soldered in a glass tube. A platinum wire coil (surface area of ca. 2 cm2) and a saturated calomel electrode, SCE, were used as the auxiliary and reference electrodes, respectively. All potentials (E) in the text are referred to the SCE. Prior to use, the working Au electrode was polished with a thin abrasive paper, washed with distilled water, and kept, in turn, in 95% ethanol (2 min) and saturated chromosulfuric acid (1 min). Afterward the electrode was rinsed with distilled water and subjected to repeated cyclic potential action in the range from 0.4 to 1.6 V at a potential scan rate (v) of 50 mV/s in 0.1 M H2SO4 until a stationary background current was established. Following the electrochemical pretreatment, the gold electrode was washed with distilled water and dried with a stream of argon gas. A thin layer (ca. 300-350 µm) of the viscous cubic MO/ VK1/H2O (64:1:35 wt %) or MO/H2O (61:39 wt %) phase was applied to the surface of the Au electrode. The layer of the cubic phase was subsequently covered with a 30-µm thick dialysis membrane, which was held in place by an O-ring. After preparation, and between experiments, the modified working electrodes were stored in a 0.1 M potassium phosphate buffer at pH 7.0. The 0.1 M potassium phosphate buffer, pH 5-8, also served as a supporting electrolyte. All electrochemical measurements were carried out under anaerobic conditions obtained by bubbling high-purity argon through the buffer for 30 min.
Results and Discussion This work is concerned with the effect of the introduction of small amounts of VK1 into liquid crystalline and reverse micellar phases of the MO/W system. The phase behavior, the swelling of the liquid crystalline phases, the mobility of the components, and the location of the VK1 in the lipid bilayers of the different structures were investigated by various techniques, and the results will be discussed in the following sections. Phase Behavior of the MO/VK1/W Tenary System. The phase behavior of the MO/W binary system has been determined by Hyde et al.24 Their results applied below are as follows: At room temperature, MO forms a reverse micellar phase (L2) at very low water content (4-5 W wt %) and a lamellar phase (LR) upon further addition of water (8-22 W wt %). In the region of 25-40 water wt % two different bicontinuous cubic structures belonging to the body-centered space group Ia3d (CG phase) and to the primitive space group Pn3m (CD phase) appear at low (35) Luzzati, V.; Mustacchi, H.; Skoulios, A.; Husson, F. Acta Crystallogr. 1960, 13, 660. (36) Hernqvist, L. Polymorphism of Fats. Ph.D. Thesis, Lund University, 1984.
and high water content, respectively. At a water content larger than 40 wt % a cubic (CD) + water region appears. In the region 20-30 water wt % a reverse hexagonal (HII) phase forms at T > 80 °C. These findings were reconfirmed by the recent work of Briggs et al.27 with the caveat that the fluid isotropic phase (L2) was observed only above 30 °C, whereas a lamellar crystalline phase (Lc) was identified below this temperature. Figure 2a shows the composition of the samples used to investigate a limited region of the ternary MO/VK1/W system at 25 °C. On the basis of visual inspection, optical microscopy (typical pictures shown in Figure 2c and d), and SAXS, the different phases were identified. From these data a schematic phase diagram, which illustrates the effect of solubilizing VK1 in the MO/W system, was constructed (Figure 2b). At a VK1 content around 1 wt % or less, the same sequence of phases, as found in the binary system, was observed. A lamellar phase and two types of cubic phases with crystallographic space groups Ia3d (CG) and Pn3m (CD) were identified by using SAXS (Table 1). Figure 2c shows an optical microscope picture of a typical lamellar sample (88.8 wt % MO, 1.1 wt % VK1, and 10.1 wt % W). In addition, the presence of VK1 up to around 1 wt % does not affect the phase transition from the CD phase to the CG phase, which occurs at about 35 water wt %. As for the binary system, the CG phase covers a much larger concentration range of MO than the CD phase. The CG phase, at high water content, and the CD phase undergo a quite drastic change upon addition of a few percent of VK1, leading to the formation of large, partially birefringent two-phase regions. A reverse hexagonal phase (HII) in equilibrium with a cubic phase (C) is likely to occur. Figure 2d shows the typical texture of the hexagonal phase (77.7 wt % MO, 3.9 wt % VK1, and 18.4 wt % W). The CD phase, containing more than 1 wt % VK1, separates into CD + water. In the border between this region and the two-phase region (HII + C), a so far uncharacterized three-phase region containing excess water appears. Moving toward lower water content, the CG and LR phases can solubilize much larger amounts of VK1 (about 5-7 wt %) before undergoing the transition to a reverse hexagonal phase. In addition the narrow isotropic fluid region (L2) seems capable of accommodating a larger amount of VK1. The exact extent of this phase region was hard to establish since it is very narrow. Thus the samples containing one phase only after 1 month of equilibration showed, after 5 months, a solid phase in equilibrium with the L2 phase. The increase of temperature, besides an expected decrease of the H-bonding strength, reduces the hydration of the polar head, with a consequent decrease of the crosssectional head group area. The configurational entropy of the alkyl chains increases simultaneously, leading to an increase of their effective volume and hence of the packing parameter. If this argument applies, then, in our system, a sufficient amount of VK1 molecules dispersed among the MO chains could increase the value of ν/al enough to form the HII phase already at room temperature. Indeed we do observe a HII phase already at room temperature in the ternary MO/VK1/W system. If we assume that VK1 will play a cosurfactant role and that the packing parameters for the components MO and VK1 are roughly additive, we can define an effective packing parameter9
ν al
( )
ν ( al) ) eff
xMO +
MO
(alν )
VK1
xMO + xVK1
xVK1 (2)
Vitamin K1 Hosted in the Monoolein-Water Phases
Langmuir, Vol. 13, No. 20, 1997 5479
Figure 2. (a) Composition of the samples used to investigate the ternary MO/VK1/W system, at 25 °C. On the basis of visual inspection, optical microscopy and SAXS, the different phases were identified, and are labeled as follows: (2) solid (S); (+) S + reverse micellar (L2); (b) reverse hexagonal (HII); (×) lamellar (LR),; (4) LR + cubic of gyroid type (CG); (0) CG; (]) HII + cubic (C); (9) cubic of diamond type (CD); (1) C + water (W); (O) HII + W. (b) Schematic phase diagram based on the data in Figure 2a. The number of phases and the microstructure of the region (Undef.) were not clearly identified. (c) Optical microscope picture (magnification 80×) of a typical lamellar sample (88.8 wt % MO, 1.1 wt % VK1, and 10.1 wt % W). (d) Typical texture of the hexagonal phase (77.7 wt % MO, 3.9 wt % VK1, and 18.4 wt % W).
where xMO and xVK1 are the mole fractions of MO and VK1, respectively. The packing parameters for LR, cubic, and HII phases are about 1.0, 1.3, and 1.7, respectively.4 If we then consider two HII samples, where sample I (84.0 wt % MO, 8.4 wt % VK1, and 7.6 wt % W S xMO ) 0.348 and xVK1 ) 0.028) is on the border to the LR phase and sample II (77.7 wt % MO, 3.9 wt % VK1 and 18.4 wt % W S xMO ) 0.175 and xVK1 ) 0.007) is on the border to the CG phase, the amphiphile in both samples is likely to have an effective packing parameter (ν/al)eff ≈ 1.7. Monoolein without VK1 in sample I would have a (ν/al)MO ≈ 1.0 and in sample II a (ν/al)MO ≈ 1.3. If we then estimate the corresponding value for VK1 by using eq 2, we end up with values of (ν/al)VK1 of 10.5 and 11.7 for samples I and II, respectively. By considering the molecular structure of VK1, these are clearly unrealistic values for an ordinary surfactant, thus suggesting that the large impact of VK1 on the phase behavior of the MO/water system cannot be explained in terms of an effective change of the packing parameter. The concentration of VK1 is simply too low. Instead we have to consider a varying lateral distribution of VK1 in the lipid bilayer. The locally higher concentration of VK1 will then trigger phase transitions. This is found in many model membrane systems, as discussed by Raudino.37 It is noteworthy that the LR phase is less sensitive to VK1 in contrast to the curved bilayer structures, i.e. the cubic phases. In the former case the lateral distribution of the solubilized VK1 is probably facilitated compared to that (37) Raudino, A. Adv. Colloid Interface Sci. 1995, 57, 229.
at a curved interface like in the cubic phases. This is especially pronounced in the CD phase, which is geometrically more constrained than the CG phase and contains circular necks which restrict the packing of the lipid bilayer.4 As a matter of fact the CD phase is more sensitive to the addtion of VK1. Moreover it is worth noting that the LR phase undergoes a transition to a HII phase in the MO/VK1/W ternary system upon increasing the amount of VK1, while a transition to a L2 phase occurs in the binary system with increasing temperature. Swelling Behavior of the Cubic and Lamellar Phases. Small angle X-ray data were used to monitor the influence of water content on the interlayer spacings of the cubic and lamellar phases. The dimensions of the unit cell change discontinuously with water content at the various phase transitions. However, if we plot the dominating spacing, i.e. (001), (211), and (110) for LR, CG, and CD, respectively, versus the water wt %, an almost linear relationship is observed (Figure 3), which has been reported earlier.4,23 This is regarded as a confirmation of the assigned values for hkl. It is important to note that this linear trend is retained in spite of the fact that the samples contain different amounts of VK1. The swelling of MO cubic phases has been extensively studied,25,38 and it has been shown that a linear swelling is just a first approximation. To describe the swelling of a bicontinuous cubic phase, Engblom and Hyde derived (38) Engblom, J.; Hyde, S. T. J. Phys. II 1995, 5, 171.
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Table 1. X-ray Diffraction Data of the Cubic and Lamellar MO/W/VK1 Phases at 25 °C composition (wt %) MO
W
VK1
dhkla (Å)
h 2 + k2 + l2
a (Å)
mean ab (Å)
space group
61.5
37.8
0.7
0.0
92.2((1.2)
Pn3m
63.6
35.4
1.0
94.3 ((0.2)
Pn3m
65.0
33.8
1.2
143.9 ((1.7)
Ia3d
66.2
32.5
1.3
143.0 ((2.4)
Ia3d
71.4
27.7
0.9
125.9 ((1.6)
Ia3d
81.0
18.1
0.9
123.5 ((2.0)
Ia3d
80.4
17.8
1.8
109.7 ((1.4)
Ia3d
79.7
17.4
2.9
109.6 ((0.75)
Ia3d
83.1
15.7
1.2
103.1 ((1.8)
Ia3d
82.3
14.8
3.0
106.5 ((2.0)
Ia3d
83.2
13.1
3.7
101.8 ((2.1)
Ia3d
82.6
12.6
4.8
86.3 (96.0)c 86.8 87.0 87.2 87.7 87.6 93.0 92.2 (98.9)c 90.7 91.9 91.0 94.2 94.5 94.4 94.4 (99.9)c 93.9 94.2 141.3 144.2 142.2 145.2 145.4 143.9 141.6 141.4 144.5 140.9 142.8 147.1 124.8 124.9 125.8 128.1 123.4 125.9 123.6 121.0 107.4 109.5 110.8 109.8 109.4 111.6 108.6 109.6 110.9 109.6 109.8 109.3 101.9 102.7 102.5 102.8 102.0 106.8 105.1 104.8 107.1 106.2 105.5 110.1 99.7 99.6 102.3 101.1 103.1 105.1 100.8 99.6 100.9 100.7
Pn3m
36.0
2 (3) 3 4 6 8 9 2 3 (4) 4 6 8 10 2 3 4 6 8 9 6 8 14 16 20 22 6 8 14 16 20 24 6 8 20 24 6 8 9 10 6 8 14 16 20 24 6 8 14 16 20 22 6 8 14 16 20 24 6 8 14 16 20 24 6 8 14 16 20 24 6 8 14 16
87.1 ((0.5)
64.0
100.5 ((0.6)
Ia3d
88.4 88.8 87.5 86.5 91.9 90.9 90.5
11.6 10.1 9.9 9.7 6.6 6.8 6.3
0.0 1.1 2.6 3.8 1.5 2.3 3.2
61.0 vs (55.4) s 50.1 vs 43.5 w 35.6 w 31.0 w 29.2 w 65.8 vs 53.2 vs (49.5) vs 45.3 vw 37.5 w 32.2 w 29.8 w 66.8 vs 54.5 vs 47.2 s (40.8) s 33.2 w 31.4 w 57.7 vs 51.0 vs 38.0 vw 36.3 vw 32.4 s 31.0 s 57.8 vs 50.0 vw 38.6 vw 35.2 w 31.9 w 30.0 w 50.9 vs 44.2 s 28.1 vw 26.2 vw 50.4 vw 44.5 vs 41.2 s 38.2 s 43.8 vs 38.7 vs 29.6 w 27.4 w 24.5 w 22.8 w 44.3 vs 38.7 vs 29.6 vw 27.4 vw 24.6 w 23.3 w 41.6 vs 36.3 s 27.4 vw 25.7 vw 22.8 vw 21.8 vw 42.9 vs 37.1 vs 28.6 vw 26.6 vw 23.6 vw 22.5 vw 40.7 vs 35.2 vs 27.3 vw 25.3 vw 23.0 vw 21.4 vw 41.1 vs 35.2 vs 27.0 vw 25.2 vw 38.8 vs 39.0 vs 38.0 vs 38.3 vs 35.8 vs 35.9 vs 35.9 vs
38.8 39.0 38.0 38.3 35.8 35.9 35.9
LR LR LR LR LR LR LR
a vs, very strong; s, strong; w, weak; vw, very weak. b Mean value and standard deviation. c The marked diffraction lines do not match the indexing and are likely to be traces of another phase in the sample. Values are not used for calculating the mean values.
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Figure 3. Interlayer spacing of the dominating X-ray diffraction lines, i.e. (001), (211), and (110) for LR (.), CG (2), and CD (b), respectively, as a function of the water content.
Figure 4. Volume fraction of lipid versus the lattice parameter (unit cell dimension) obtained by SAXS of the CG phase. The solid line represents a fit of the experimental data to eq 3. For details, see text.
an expression (eq 3) which relates the volume fraction of the lipid, Φ, with the size of the unit cell (lattice parameter), a:
Φ)
c1 c2 + a a3
(3)
where
c1 )
νs(-16πχH2)1/3 Ω(t)
and c2 )
4πχνst2 Ω(t)
and t is the distance from the midplane of the bilayer to the neutral surface. This surface is defined in such a way that its area does not change upon bending. The lipid has the cross-sectional area Ω(t) at the neutral surface. The molecular volume of the lipid, νs, which can be calculated from the density and from the molecular weight, is 628 Å3 for monoolein (MW ) 357 g/mol, F ) 0.942). The homogeneity index, H, and the Euler-Poincare´ characteristics per unit cell, χ, are 0.7665 and -8 for the gyroid IPMS. Thus, by fitting the volume fraction of lipid versus the lattice parameter to eq 3, the parameters c1 and c2 are obtained. This is shown in Figure 4. Hence, an area Ω(t) ) 6.8 Å2, at t ) 10.0 Å, is calculated, which is close to the values found by Chung and Caffrey25 and Engblom and Hyde38 for the pure MO/W system. These findings strongly suggest that, unlike its effect on the phase boundaries, the addition of a small amount of VK1 does not substantially affect the swelling behavior of the CG phase. Further, for the LR phase at about 10 water wt %, the interlayer spacing does not change when increasing the VK1 content
Figure 5. NMR self-diffusion coefficients at 25 °C in samples containing 0-5 wt % VK1, shown as a function of the lipid volume fraction, Φ. DMO and DVK self-diffusion coefficients were measured in the CD, CG, and L2 phases, while Dw was measured in the CD and CG phases only. (a) Reduced water self-diffusion coefficients (Dw/D0), D0 ) 2.29 × 10-9 m2 s-1 is the water bulk value at 25 °C. The continuous line refers to the fitting of the experimental data to the linear relation Dw/D° ) (0.71-0.72)Φ (r ) 0.95); the dotted line represents the approximated equation relative to the ICR model Dw/D° ) (0.636-0.264)Φ.41 (b) Selfdiffusion coefficients of MO (DMO) (b, O, ]) and VK1 (DVK) (2). The values of DMO recorded in samples containing 0, >0 and
