J. Phys. Chem. B 1998, 102, 11035-11042
11035
Stereochemistry-Dependent Self-Assembly in Synthetic Glycolipid/Water Systems: The Aqueous Phase Structure of 1,3-Di-O-dodecyl-2-(β-maltoheptaosyl)glycerol Masakatsu Hato,* Hiroyuki Minamikawa, and Joan B. Seguer† Surface Engineering Laboratory, National Institute of Materials and Chemical Research, 1-1, Higashi, Tsukuba, Ibaraki-305-8565, Japan ReceiVed: October 14, 1997; In Final Form: October 13, 1998
We have investigated the temperature-concentration dependent phase diagram of an aqueous maltoheptaosecontaining lipid, 1,3-di-O-dodecyl-2-(β-maltoheptaosyl)glycerol, Mal7(C12)2. In the region examined (25-90 °C and 0-65 wt % lipid), two liquid crystalline phases form: a normal hexagonal phase (HI) above 40-45 wt % lipid and a second phase (most probably a lamellar phase LR) at higher temperatures (>55 °C) and higher concentrations (>60 wt % lipid). From the dilute solution to about 30 wt % lipid, an optically isotropic solution region is found where normal micelles exist. The present results together with the phase behavior of MalN(C12)2/water systems with N ) 1-5 (Hato, M.; Minamikawa, H. Langmuir 1996, 12, 1658) indicate that a preferred phase of the MalN(C12)2 where the headgroup is composed of N glucose residues linked via R-1,4O-glycosidic bonds shifts from an HII to an HI via an LR phase as N increases. The observed phase sequence makes a marked contrast to the CelN(C12)2/water systems where the headgroup is composed of N glucose residues linked via β-1,4-O-glycosidic bonds (Hato, M.; Minamikawa, H. Langmuir 1996, 12, 1658). The stereochemistry-dependent phase behavior can be interpreted in terms of different conformations of the headgroups, i.e., a “helical” conformation of the maltooligosaccharide headgroups, and an “extended” conformation of the cellooligosaccharide headgroups.
Introduction Glycolipids, amphiphiles that bear oligosaccharides as their hydrophilic headgroups, are progressively gaining importance both scientifically and technically.1-8 In biological cell membranes, glycolipids are believed to be involved in a variety of physiological events1,2 such as molecular recognition at cell surfaces4,9,10 and stabilization of the membrane structures of archaebacteria that grow under extreme environmental conditions.11 Moreover, because glycolipids can be synthesized from renewable resources such as oligosaccharides and fatty alcohols, they appear to be less environmentally damaging than many other synthetic surfactants.6 Because oligosaccharides are more hydrophilic than the oligoethyleneoxide headgroups,12,13 glycolipids are of great interest as a new type of surfactants for liposomal drug delivery14 and various aspects of biotechnology.15,16 Despite the growing attention paid to glycolipids, their physical studies have been limited. This is in part due to the difficulty in obtaining sufficient amount of chemically pure compounds either from natural membrane extracts or by synthetic methods. Though the synthetic approach has proved promising in preparing well-defined compounds,17-21 the procedures to obtain stereochemically pure compounds are still so demanding that the kinds of oligosaccharide headgroups so far synthesized and investigated have been mainly mono- and disaccharides. It is well established that molecular packing of lipid molecules in aqueous media is determined by competing interactions of * Corresponding author. E-mail:
[email protected]. Fax: 81-298-546232. † Present address: Laboratorios Miret S.A. C/Geminis, 4 Polı´gono Industrial Can Parellada, Barcelona, Spain.
the polar headgroups and the alkyl chains.22-25 Oligosaccharide headgroups can assume a variety of conformations arising from multiple choices of isomeric linkages that join the monosaccharide groups.26 Therefore, the stereochemistry of the headgroups is expected to be most relevant to the physical properties of glycolipid/water systems. Though there are several indications that isomerism of the carbohydrate headgroups determines the physical properties of alkyl glucosides27-29 and alkyl-thiotalopyranosides,30 little is known about the correlation between the headgroup stereochemistry and the phase behavior of the glycolipid/water systems, which is critical to a quantitative understanding of the molecular assembly of the glycolipid/water systems. Unexpectedly profound effects were found when we investigated a correlation between the stereochemistry of oligosaccharide headgroups and the phase behavior of aqueous glycolipids: 1,3-di-O-dodecyl-2-O-(β-glycosyl)glycerols bearing a series of cello- or maltooligosaccharides as their headgroups and phytanyl-chained glycolipids.31-33 For the cellooligosaccharide-containing lipids, CelN(C12)2 (derived from cellulose where N glucose units are linked via β-1,4-O-glycosidic bonds), an increasing value of N raises the hydrated-solid/liquid-crystalline phase transition temperature Tm. In particular, Tm jumps from 59 °C (N ) 4) to above 160 °C (N ) 5); Cel5(C12)2 decomposes above 170 °C prior to melting. Therefore, Cel5(C12)2 can no more form any liquid crystalline phase and is practically insoluble in water. On the other hand, the Tm of the maltooligosaccharide-containing lipids, MalN(C12)2 (derived from amylose where N glucose units are linked via R-1,4-O-glycosidic bonds), decreases as N increases. With N ) 2 or 3, the phase behavior of both the CelN(C12)2/water and MalN(C12)2/water systems do not differ significantly: an LR phase + water dispersion in a dilute solution regime and an LR
10.1021/jp9733328 CCC: $15.00 © 1998 American Chemical Society Published on Web 12/08/1998
11036 J. Phys. Chem. B, Vol. 102, No. 52, 1998
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Figure 1. Chemical structure of 1,3-di-O-dodecyl-2-O-(β-maltoheptaosyl)glycerol, Mal7(C12)2. The headgroup is composed of 7 glucose residues linked via R-1,4-O-glycosidic bonds.
phase at higher lipid concentrations. Distinct phase behavior that clearly reflects the stereochemistry of the headgroups becomes apparent when N is larger than 4. As opposed to a solid + water dispersion of the Cel5(C12)2/water system, the Mal5(C12)2/water system exhibits a nearly isotropic solution (but still not a single phase) at high dilution, strongly suggesting that the aqueous MalN(C12)2 would form an HI phase and normal micelles when a value of N is sufficiently large. However, the conclusive evidence was still lacking since we could detect neither an HI phase nor a normal micellar solution for the MalN(C12)2/water systems so far investigated (up to N ) 5). Furthermore, as interoligosaccharide interactions are not well understood at present, extrapolation of the observed phenomena into an unexplored regime may not be justified. This prompts us to extend our earlier work31-33 and to investigate a phase diagram of the maltoheptaose-containing lipid Mal7(C12)2/water system. The major objective of this work is to obtain direct evidence in support of the above hypothesis by confirming an HI and a normal micellar solution in the Mal7(C12)2/water system. Finally, the possible molecular mechanism of the stereochemistry-dependent phase behavior will be discussed. Experimental Section Materials. The chemical structure of Mal7(C12)2 used in this study is shown in Figure 1. The glycolipid was synthesized by the same method reported in the previous paper.34 We coupled a chromatographically purified oligosaccharide (Seikagaku Kogyo, Tokyo; >97%) β-glycosidically with a hydrophobic group, 1,3-di-O-dodecylglycerol which was prepared from purest grade dodecanol (Tokyo Kasei Kogyo; >99.5% by GC) and epichlorohydrin.34,35 All reactions were carried out under dry conditions. Elemental analysis for carbon and hydrogen atoms gave satisfactory agreement with calculated values assuming two moles of hydrated water. (Found: C, 51.57; H, 8.15. Calcd for C69H126O382H2O: C, 51.81; H, 8.19.) The purity of the lipid estimated by NMR and TLC is at least 95%. Other lipids were the same compounds described in the previous papers.31,32,34 n-Dodecyl-β-maltoside was obtained from Wako Pure Chemicals Co. and used as received. There was no minimum in the surface tension curve and the value of cmc agreeed well with the literature value36 (see Figure 3). Water was purified as described in the previous paper, and the purity was successively checked by surface tension measurement (72.4 mN/m at 22 °C).37 Differential Scanning Calorimetry (DSC). Calorimetric measurement of the aqueous glycolipid was performed with a Seiko DSC120 differential scanning calorimeter over the temperature range from - 50 to 100 °C. An aqueous lipid sample of known composition was sealed in a silver DSC capsule. The capsule was then taken repeatedly through heating-cooling cycles between 0 and 100 °C to ensure complete hydration of the lipid.38 We confirmed that silica gel thin-layer
Figure 2. The DSC thermograms of the aqueous Mal7(C12)2. (a) 45 wt % lipid, close to the phase boundary of the HI phase. (b) 55% lipid (one-phase region of an HI phase). The vertical line in the figure corresponds to 0.005 cal/K.
Figure 3. Surface tension of (a) aqueous Mal7(C12)2 at 25 °C. For comparison surface tension of (b) n-dodecyl-β-D-maltoside is also plotted.
chromatography of the lipid exhibited no sign of decomposition after this pretreatment. We equilibrated the sample at -50 °C for 3 h before a heating run was initiated. Since values of Tm are close to 0 °C, DSC measurements were performed with samples of varying lipid concentrations (see later). Because the thermodynamic parameters obtained at the heating rates of 0.5 °C/min and at 1.0 °C/min agreed well with each other, we mainly employed a rate of 1.0 °C/min. X-ray Diffraction. Samples of known concentrations were prepared by weighing the desired amount of lipid and pure water in a glass cell, and the samples were treated with repeated cycles of freezing and thawing. The aqueous lipid was then filled into a thin-walled quartz capillary (1.5 mm in diameter, GLAS, Berlin), and the capillary was flame-sealed. The sample was inserted into a Mettler HP82HT sample holder thermostated to an accuracy of (1 °C and exposed to a pinhole collimated X-ray beam (0.3 mm in diameter) for 30 min to 7 h. A Rigaku Rotaflex generator RU-200 was served as the source for nickel-filtered Cu KR radiation (λ ) 0.1542 nm, 40 kV, and 100 mA). The lipid concentrations examined were 25, 30, 39,42,45, 47, 53, 58, 61, and 64 wt %. The measurements were carried out at 25, 35, 45, 55, 65, 75, and 90 °C in this sequence. At each temperature, small-angle X-ray scattering (SAXS) measurements were performed after three different incubation periods of 30, 60, and 120 min. We confirmed that these three measurements yielded identical diffractograms. After the measurement at the highest temperature, 90 °C, the sample temperature was lowered down to 25 °C, where a final SAXS measurement was performed. We confirmed the second measurement at 25 °C yielded an identical
Stereochemistry-Dependent Self-Assembly diffraction to that of the initial measurement at 25 °C. At 25 °C, we also monitored the change in the diffractograms over 1 week without noting any notable change both in the peak positions and the intensity, an indication that the thermal equilibrium of the present systems is fairly rapidly reached at least at 25 °C. X-ray diffraction patterns were recorded with an imaging plate (Fuji Film Ltd., HR-III N) in a flat camera (the sample to film distance ) 300 mm), and were digitally read on a Rigaku RINT2000 system. TLC after SAXS measurements did not exhibit any sign of decomposition of the lipids. Optical Microscopic Observation. The lipid/water system was examined with an Olympus BHS-751-P polarizing microscope equipped with a Mettler FP82HT hot stage thermostated to an accuracy of (1 °C. Microscope slides were buffed with lint-free paper tissue immediately before use. A small amount of the aqueous lipid was transferred with a pipet from a glass vial onto a glass slide and was immediately covered with a slide cover. The sample was then sheared between the slide and the cover to a thickness of about 5-10 µm and was left for a few minutes on the constant temperature stage for relaxation and thermal equilibration.39 The different liquid crystalline phases were optically identified according to the birefringent textures observed between crossed polarizers.40,41 Surface Tension and Surface Pressure. The surface tension of the aqueous Mal7(C12)2 was measured by using a Kyowa Kaimenkagaku Model CBVP-A3 surface tensiometer (a Whilhelmy plate technique) with a sand-blasted platinum plate as a pressure probe under a saturated water vapor condition. The surface tension was measured as a function of time until the time dependence (dγ/dt) reached at least a value less than 0.1 mN/m h (0.1 mN/m is a detection limit of the apparatus). The experimental temperature was 25 ( 0.1 °C. The surface pressure-area isotherm of Mal7(C12)2 was determined at 8 ( 0.2 °C by a Wilhelmy technique using a sand-blasted platinum plate. The monolayer mass density was varied by means of a successive addition method.32 The surface pressure was measured as a function of time until the time dependence (dΠ/dt) reached at least 10-3 mN/m s at which point we took the measured Π to be near equilibrium. More detailed procedures were described in the previous manuscript.32,37
J. Phys. Chem. B, Vol. 102, No. 52, 1998 11037 a
b
Results DSC. DSC measurements were performed with samples of varying lipid concentrations. As typical examples, Figure 2 shows DSC thermograms of 45 and 55 wt % lipid over the temperature range -50 to 30 °C. We could detect neither an endothermic nor exothermic peak in the range of 30-100 °C. At 35 wt % lipid (data not shown), a two-phase region of an HI and an isotropic phase (see below for the phase characterization and Figure 6), a large endothermic peak due to melting of ice with a peak temperature at 0 °C overwhelms a thermogram. We can see the hydrated solid to liquid crystalline phase transition of the lipid only from a broad shoulder at around -7 to -9 °C. At 45 wt % lipid (curve a), close to the phase boundary of the HI phase, a peak due to melting of frozen aqueous phase significantly reduces and a shoulder becomes more evident with a peak temperature at about -6 to -7 °C. At 55 wt % lipid (curve b), one-phase region of an HI phase, a thermogram exhibits only one endothermic peak at -6 °C that is associated with the hydrated solid to liquid crystalline phase transition of the lipid. In a separate experiment of 50 wt % lipid, a one-phase region of an HI phase, we confirmed that the thermogram also exhibits only one endothermic peak with a same peak temperature at -6 °C (data not shown). We here
Figure 4. (a) SAXS diffraction patterns of the 53 wt % aqueous Mal7(C12)2 at different temperatures. All the patterns between 25 °C and 90 °C are characterized by four sharp peaks with spacings in the ratio of d1:d2:d3:d4 ) 1:1/x3:1/2:1/x7, which is consistent with an HI phase. q ) 4π sin θ/λ is the scattering vector, where 2θ is the scattering angle. (b) SAXS diffraction patterns of the 61 wt % aqueous Mal7(C12)2 at different temperatures. The patterns up to 45 °C are consistent with an HI phase, while those above 60 °C, sharp diffraction peaks in the ratio of d1:d2 ) 1:1/2, characteristic of a layered structure, start to predominate. At 55 °C, the HI phase appears to coexist with the layered structure. q ) 4π sin θ/λ is the scattering vector, where 2θ is the scattering angle.
11038 J. Phys. Chem. B, Vol. 102, No. 52, 1998
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TABLE 1: Small-Angle X-ray Scattering Data of the Mal7(C12)2/Water System lipid concentration (wt %)
temperature (°C)
53
25 90 25 65 90
61
a
Bragg’s d-spacings (nm) d1
d2
d3
d4
7.28, 4.23, 3.67, 2.75 6.97, 4.04, 3.51, 2.61 7.09, 4.07, 3.55, 2.71 6.13, 3.11 6.07, 3.04
d-spacing ratio d1:d2:d3:d4
type of liquid crystalline phase
1.0:0.581:0.504:0.378 1.0:0.580:0.504:0.374 1.0:0.574:0.501:0.382 1.0:0.507 1.0:0.501
HI HI HI a a
Tentatively assigned as an LR phase.
Figure 6. A partial phase diagram of the Mal7(C12)2/water system up to 65 wt % lipid. 2φ: an HI + isotropic solution coexisting region.
Figure 5. Birefringent textures of the 64 wt % aqueous Mal7(C12)2 at different temperatures (×100). (a) A “nongeometrical” texture at 25 °C, suggesting an HI phase. (b) An “oily streak” texture at 65 °C, suggesting an LR phase.
take a peak temperature of a first isolated peak (an isolated peak first appeared when the lipid concentration was increased), -6 °C, as a value of Tm of Mal7(C12)2. Values of Tm of Mal6(C12)2 and Mal5(C12)231 listed in Table 2 were obtained in a similar manner, Clipid ≈ 60 and 70 wt % for N ) 6 and 5, respectively. Thus, except for an extremely concentrated lipid regime, the aqueous Mal7(C12)2 is in a fluid state above 0 °C. A broad X-ray diffraction peak observed at about 0.46 nm also supports this conclusion. The enthalpy change ∆H and the entropy change ∆S associated with the phase transition are 8 kcal/mol and 30 cal/mol K, respectively. Surface Tension and Cmc. Figure 3 shows the surface tension (γ) vs concentration curve of the aqueous Mal7(C12)2 together with that of n-dodecyl-β-maltoside. The surface tension of the aqueous Mal7(C12)2 decreases with the lipid concentration Clipid and attains a constant value of 36 ( 0.5 mN/m above Clipid ) 5 × 10-6 M. No minimum in the γ vs log Clipid curve indicates the absence of surface active impurities. According to the Gibbs equation, the constant value of the surface tension implies that the activity of the lipid is practically constant above
Clipid ) 5 × 10-6 M. This indicates that a “phase-separation” like phenomenon (micelle formation) occurs at least above 5 × 10-6 M.42 It is, however, noted that the slope of the γ vs log Clipid curve below cmc is unusually large; the Gibbs equation gives an unrealistically small cross section area of Mal7(C12)2, ∼ 0.3 nm2/molecule, compared with that of n-dodecyl-βmaltoside, 0.5 nm2/molecule. This is due to an experimental problem that the γ values measured below cmc were not thermodynamic equilibrium values, presumably owing to very long equilibrium times in a lower concentration regime (more than 30 h) and/or to contact angle hysteresis at the platinum plate-solution-air interface. The deviations from equilibrium values increase as Clipid decreases. At 2 × 10-6 M, for example, the measured γ never lowered below ∼60 mN/m even after 80 h of equilibration. The value of 60 mN/m is erroneously high compared with a value extrapolated from the surface tension curve measured in the higher concentration range. Thus, 5 × 10-6 M may be considered to be an upper limit of the cmc. More detailed discussion of this subject will be described in a separate paper.43 Small-Angle X-ray Diffraction and Optical Microscopic Observations of the Liquid Crystalline Phases. The results of SAXS experiments can be represented in the two regions of the phase diagram, and the relevant numerical values are listed in Table 1. The diffraction patterns between about 30 and 60 wt % lipid are exemplified in Figure 4a, where a sequence of the diffraction patterns of 53 wt % lipid at different temperatures is shown. All the patterns between 25 and 90 °C are characterized by four sharp peaks with the ratio d1:d2:d3:d4 ) 1:1/x3:1/2:1/x7, which is consistent with parallel cylinders packed in a two-dimensional hexagonal lattice. In the digitally recorded diffractograms, the fourth-order diffraction was detected as a complete ring by adjusting the image contrast (data not shown). The values of first-order diffraction d1 are rather temperature insensitive: 7.3 nm at 25 °C and 7.0 nm at 90 °C. The patterns of the two-dimensional hexagonal lattice persist-
Stereochemistry-Dependent Self-Assembly
J. Phys. Chem. B, Vol. 102, No. 52, 1998 11039
TABLE 2. Molecular Parameters of MalN(C12)2 and CelN(C12)2 and the Phase Behavior of the Corresponding Lipid/Water Systems compound
A* d,e
a
0.40
Glc(C12)2
Shcd,f
AMM2d,g 0.4
phase behavior in a dilute lipid concentration regime (T > Tm, C e 30 wt %)
Tmh (°C) 50
refs
an isotropic phase + water (∼70 °C)
31
LR + water LR + water a lipid aggregate + water normal micelles/HI
31, 32 31, 32 31, 32 this work this work
LR + water LR + water LR + water a (hydrated) crystal + water
31, 32 31, 32 31, 32 31, 32
MalN(C12)2b Mal2(C12)2 Mal3(C12)2 Mal5(C12)2 Mal6(C12)2 Mal7(C12)2
0.48 0.55 0.64 0.67 0.72
0.8
0.4 0.6 1.2 1.25 1.3
45 15 -3 -3 -6 CelN(C12)2c
Cel2(C12)2 Cel3(C12)2 Cel4(C12)2 Cel5(C12)2
0.40 0.40 0.40
0.4 0.4 0.4 0.4
46 66 59 >160
a Glc(C12)2 ) Mal1(C12)2 ) Cel1(C12)2. b MalN ) a maltooligosaccharide headgroup where N glucose residues are linked via R-1,4-O-glycosidic bonds. c CelN ) a cellooligosaccharide headgroup where N glucose residues are linked via β-1,4-O-glycosidic bonds. d In nm2/molecule. e A*: a fully compressed molecular area (at Π ≈ 40 mN/m) obtained from the Π-A isotherm. f Shc: a molecular cross section area at the hydrophobic core-headgroup interface in a liquid crystalline phase. g AMM2: a cross section area of the minimum energy conformation of an oligosaccharide headgroup derived from Allinger’s MM2 calculation in a vacuum. h Tm: hydrated solid-liquid crystalline phase transition temperature.
down to about 30 wt %, below which the solution becomes isotropic and we could no longer detect the diffraction peaks of the hexagonal phase even after 7 h of exposure. Between about 30 and 45 wt %, diffractions exhibit the hexagonal phase with a constant value of d1, an indication of a hexagonal phase + isotropic solution coexisting region. In the isotropic solution, oil soluble dye methyl yellow was solubilized as expected. This together with the surface tension result indicate that the isotropic region is a normal micellar solution. By analogy with other lipid/ water systems,44 the structure of this hexagonal phase is identified as type HI (a normal hexagonal phase). The diffraction patterns above 60 wt % lipid are exemplified in Figure 4b where a sequence of the diffraction patterns of 61 wt % lipid at different temperatures is shown. The patterns up to 45 °C are consistent with the HI phase, while sharp diffraction peaks in the ratio d1:d2 ) 1:1/2 suggest a layered structure starting to predominate above 60 °C. At 55 °C, the diffractions due to the HI phase (indicated by arrows) appear to coexist with the layered structure. The repeat distance d1 for the layered structure is also temperature insensitive: 6.13 nm at 65 °C and 6.07 nm at 90 °C. Birefringent textures of 64 wt % lipid as observed with the polarizing microscope at different temperatures are shown in Figure 5. The texture in the HI phase region (25 °C) exhibits a “nongeometric texture”.40 At 65 °C, the texture turns to a characteristic “oily streak” structure of a lamellar phase (LR).40 Taken together, the aqueous structure at higher temperatures may be tentatively assigned as an LR phase. Partial Phase Diagram of the Mal7(C12)2/Water System. A partial phase diagram of the Mal7(C12)2/water system up to 64 wt % lipid which is determined mainly by SAXS measurements is shown in Figure 6. Owing to the limited amount of the lipid, we measured samples of discrete concentrations, i.e., 25, 30, 39, 42, 45, 47, 53, 58, 61, and 64 wt %. The temperatures examined are 25, 35, 45, 55, 65, 75, and 90 °C for each composition. As the present phase diagram was constructed from these data-matrixes, the uncertainty in the phase boundary is expected to be (3 wt % and (3 °C, respectively. In the region examined, the two liquid crystalline phases form in the temperature range 25-90 °C. A normal hexagonal phase (HI) starts to form above 40-45 wt %. At higher temperatures (above
Figure 7. Pressure-area isotherms of Mal7(C12)2 (O) and Mal5(C12)2 (b). The collapse pressures are about 42-43 mN/m for both cases.
55 °C) and concentrations (above ∼60 wt %) another phase (tentatively assigned as a lamellar phase, LR) emerges. From the dilute solution to approximately 30 wt %, an optically isotropic micellar solution region is found where the viscosity increases with increasing Mal7(C12)2 concentration. Clouding phenomena which are characteristic of conventional nonionic surfactants bearing polyoxyethylene groups were not observed at least up to 90 °C. As mentioned above, the diffractions due to the HI phase measured between about 30 and 45 wt % give a constant value of d1, an indication of an HI + isotropic solution coexisting region. It is also noted that the phase boundaries are temperature insensitive, suggesting that the hydration of the headgroup does not change significantly with temperature. These results clearly indicate that Mal7(C12)2/water system forms normal micelles and an HI phase. It is finally noted that though the mesophase structures identified in the present study were an HI and an LR phase, the presence of other structures such as a cubic phase is not completely excluded. Pressure-Area Isotherm. The Π-A isotherm measured up to a film collapse pressure for the Mal7(C12)2 is compared with that of Mal5(C12)2 in Figure 7. The isotherms are of an expanded type, and Mal7(C12)2 gives a more expanded isotherm than Mal5(C12)2. The isotherm of Mal6(C12)2 exists between Mal5(C12)2 and Mal7(C12)2 (data not shown). From the results, we estimate fully compressed headgroup areas (A*) of 0.64, 0.67, and 0.72
11040 J. Phys. Chem. B, Vol. 102, No. 52, 1998 nm2 for N ) 5, 6 and 7, respectively. This is in agreement with the trend observed for the shorter maltooligosaccharide-containing lipids, MalN(C12)2 with N ) 1-4 (see Table 2). Thus we can conclude that the molecular cross section area of MalN(C12)2 increases as N increases. Finally, it has to be emphasized that the isotherms, even that of micelle-forming Mal7(C12)2, are stable and reversible, so that we could obtain reliable information of the molecular cross section areas of the lipids A*. We monitored the film pressures over 10-12 min at each surface molecular density and confirmed that a change in the film pressure was not more than 0.1 mN/m (
