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Effect of Solvent Dielectric Constant on the Formation of Large Flat Bilayer Stacks in a Lecithin/Hexadecanol Hydrogel Yasuharu Nakagawa,*,† Hiromitsu Nakazawa,‡ and Satoru Kato*,‡ †

Beauty Care Laboratory, Kracie Home Products, Ltd., 134, Goudo-cho, Hodogaya-ku, Yokohama, Kanagawa 240-0005, Japan Department of Physics, School of Science & Technology, Kwansei Gakuin University, 2-1, Gakuen, Sanda, Hyogo 669-1337, Japan

‡

S Supporting Information *

ABSTRACT: We investigated the effect of dielectric properties of the aqueous medium on the novel type of hydrogel composed of a crude lecithin mixture (PC70) and hexadecanol (HD), in which charged sheet-like bilayers are kept far apart due to interbilayer repulsive interaction. We used dipropylene glycol (DPG) as a modifier of the dielectric properties and examined its effect on the hydrogel by synchrotron X-ray diffraction, differential scanning calorimetry (DSC), polarized optical microscopy, and freeze-fracture electron microscopy. We found that at a DPG weight fraction in the aqueous medium WDPG ≈ 0.4, the bilayer organization is transformed into unusually large flat bilayer stacks with a regular lamellar spacing of 6.25 nm and consequently disintegration of the hydrogel takes place. Semiquantitative calculation of the interbilayer interaction energy based on the Deyaguin−Landau− Verwey−Overbeek (DLVO) theory suggested that the reduction of the aqueous medium dielectric constant ε by DPG may lower the energy barrier preventing flat bilayers from coming closer together. We inferred that the size of the bilayer sheet increases because the reduction of ε promotes protonation of acidic lipids that work as edge-capping molecules.

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INTRODUCTION Hydrogels formed in ternary systems of a surfactant, fatty alcohol, and water are widely used for the formulations of skin moisturizing creams1−5 and hair conditioners6,7 in the cosmetic and pharmaceutical industries. These ternary systems have been characterized by various techniques such as X-ray diffraction,1,3 light and electron microscopy,8−12 differential scanning calorimetry,2,5,13,14 rheometry,2,4,8,11,13,15 conductivity and dielectric analyses,16−20 NMR,21 and laser Raman spectroscopy.22 In these systems, the gelation is induced by formation of a viscoelastic network (convoluted bilayer network) throughout the solution. Recently, we found that the ternary system of the crude soybean lecithin mixture PC70, hexadecanol (HD), and water forms a novel type of hydrogel that consists of sheet-like bilayers with open edges, and proposed a new gelation mechanism that water continuum surrounding homogeneously distributed charged bilayer sheets works as the structural network in the PC70/HD/water gel.23,24 We speculated that interbilayer repulsion based on the Gouy−Chapman electric double layer theory25 is stronger than the van der Waals attractive force, and consequently the sheet-like bilayers keep the interbilayer distance as far as possible to make their distribution in the solution homogeneous. In order to fully understand the formation mechanism of the PC70/HD hydrogel and establish a method to produce a stable gel, we need to clarify the solvent effect on the gelation. In this study, we expected that the physicochemical properties and/or © 2016 American Chemical Society

molecular organization of the hydrogel would be changed by the addition of water-miscible molecules with low dielectric constants such as alcohol and polyol because these molecules modify the electric double layer repulsion between charged bilayers. Ethanol, having a relatively low dielectric constant, is one of the most well-known water-miscible molecules to change the water polarity, leading to the change in the physicochemical properties of the hydrogel. However, lower alcohols such as methanol, ethanol, and propanol are known to dissolve into the lipid bilayer and transform the normal bilayer structure into the interdigitated structure.26−32 Therefore, lower alcohols are not suitable for our purpose of clearly revealing the solvent effect (dielectricity effect) on the gelation. On the other hand, polyols with relatively high dielectric constants are supposed to have little impact on the electric double layer repulsion between bilayers. In fact, preliminary experiments showed that glycerol and sorbitol had no critical effect on the molecular organization and the chain-melting transition temperature of the PC70/HD hydrogel. Hence, we focused on dipropylene glycol (DPG), a polyol with a low dielectric constant similar to ethanol, as a strong modifier of the interbilayer interaction through variation in solvent properties. We systematically investigated the effect of DPG on the PC70/ Received: March 29, 2016 Revised: June 1, 2016 Published: June 20, 2016 6806

DOI: 10.1021/acs.langmuir.6b01217 Langmuir 2016, 32, 6806−6814

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Langmuir HD hydrogel by synchrotron X-ray diffraction, differential scanning calorimetry (DSC), polarized optical microscopy, and freeze-fracture electron microscopy. The addition of DPG to the PC70/HD/water system induced formation of unusually large flat bilayer stacks (FBS) and consequently disintegration of the hydrogel. We will discuss the mechanism of the FBS formation on the basis of the influence of the change in the solvent dielectric constant on the interbilayer interactions to provide new insights into regulation of the hydrogel formation.

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Figure 1. Photograph of PC70/HD solutions containing increasing concentration of DPG. The lipid (PC70 + HD) concentration was 5 wt %. The samples between DPG = 0 and 30 wt % look like homogeneous hydrogel whereas the samples at DPG = 40 and 50 wt % are obviously inhomogeneous. Syneresis was observed in these inhomogeneous solutions after standing for a few months.

EXPERIMENTAL SECTION

Sample Preparation. Hydrogenated soybean lecithin (PC70), hexadecanol (HD), and dipropylene glycol (DPG) were purchased from Nippon Fine Chemical Co., Ltd., Sigma−Aldrich Co. and Adeka Co., respectively. Other reagents used were of analytical grades. All materials were used without further purification. Samples were prepared as follows: All lipids (PC70 and HD) were dissolved in chloroform/methanol (2:1, v/v) to be mixed in the weight ratio of PC70/HD = 3:2 at 40 °C. The solvent was first removed with a rotary evaporator, and subsequently with a high vacuum pump for about 2 h. The remaining film was dispersed in the mixed solution of purified water and DPG in a desired weight ratio, vortexed at 80 °C for 5 min, and cooled down to 30 °C with gentle vortexing. This operation was repeated several times to fully hydrate the sample. The final lipid concentration was 50 mg/mL. We performed all experiments in this work at a fixed PC70/HD weight ratio (PC70/HD = 3:2), which we previously found is suitable for forming a homogeneous hydrogel.23 Polarization Microscopy. The macroscopic morphology of the bilayers was observed with a Nikon E600 Pol polarizing microscope under open nicols, and the molecular orientation was checked under crossed nicols. The sample solution was observed without dilution. Freeze-Fracture Electron Microscopy. The specimen frozen rapidly in liquid nitrogen slush was placed in a freeze-fracture apparatus (JFD-9010, JEOL Ltd.), and a replica film for the fractured surface was obtained according to a conventional method described elsewhere.33 The replicas were observed with a transmission electron microscope (JEM-1400, JEOL Ltd.) operating at 100 kV. X-ray Diffraction. Synchrotron X-ray diffraction experiments were performed at Station BL40B2 of SPring-8, Japan. An aliquot of specimen solution was sealed into a thin glass capillary with a diameter of about 1 mm and equilibrated at the room temperature before the measurement. The two-dimensional diffraction pattern was recorded with an imaging plate detector (R-AXIS VII, Rigaku). The wavelength λ was 0.08857 nm, and the exposure time was 30 s. The camera length was set to be about 500 mm so as to be appropriate for simultaneous detection of small and wide-angle diffraction and calibrated using cholesterol powder crystals. One-dimensional intensity profiles as a function of the modulus of scattering vector s = 2sin θ/λ (2θ is the scattering angle) were obtained by integrating the two-dimensional diffraction patterns along the azimuthal direction and divided by 2πr, where r is the distance from the beam center. Differential Scanning Calorimetry. The effect of DPG on the phase behavior of the PC70/HD mixed bilayers was examined by differential scanning calorimetry (DSC). The sample solution was loaded into an aluminum pan and set in a DSC apparatus (DSC6220, SII Nano Technology Inc.). The temperature was increased from 15 to 85 °C at the scanning rate of 5 K/min. For convenience, the peak temperatures in the DSC thermograms were measured for analysis of the DPG-induced change in the transition temperature Tm.

solutions with 0−30 wt % DPG are homogeneous, the sample solutions with 40 and 50 wt % DPG are evidently inhomogeneous. In addition, syneresis was observed in these inhomogeneous solutions after standing for a few months. Thus, the addition of a high concentration of DPG induced disintegration of the hydrogel composed of PC70 and HD. DPG-Induced Change in Bilayer Morphology and Organization. We examined the DPG-induced change in the bilayer organization in the PC70/HD bilayer system by polarized optical microscopy and freeze-fracture electron microscopy (Figures 2, 3). In the absence of DPG, no clear structure was seen in the optical micrographs (Figures 2a, 2b) and the bilayers had an amorphous surface morphology and no interbilayer coherence (Figure 3a). The fact that no orientational birefringence was observed in Figure 2b indicates that the orientation of the flat bilayers in the light path is isotropic on average. In contrast, a number of bright lines with strong anisotropy were seen in the presence of 40 wt % DPG (Figure 2c, 2d). The lines, which are likely to be parallel to the bilayer surface, were brightest at 45° from the polarization axis of the polarizer when we rotated the sample. This means that the long axis of the lipid molecules is oriented parallel to the bilayer normal. Electron microscopic observation was consistent with these results (Figure 3b): Flat sheet-like bilayers with a width of more than 10 μm were stacked to form a multilamellar assembly. These results suggest that the bright straight lines observed in the optical micrographs should represent the cross section almost normal to the flat sheet-like bilayers. If it is the case, the stacked bilayer assembly has an end-to-end distance of 10−100 μm and a thickness on the order of 1 μm. Formation of large sheet-like bilayer stacks in the presence of 40 wt % DPG was further supported by the two-dimensional Xray diffraction pattern, in which the reflection intensity at 0.159 nm−1 was inhomogeneous in the circular direction (arrows in Figure 4). This means that the orientation of flat membranes is concentrated in a particular direction. These results are consistent with the formation of large flat bilayer stacks, which cannot orient randomly in the range of beam size (200 μm × 200 μm). DPG-Induced Change in Lamellarity. We investigated the mechanism of the DPG-induced disintegration of the hydrogel in the PC70/HD bilayer system by synchrotron X-ray diffraction measurements. Figure 5 shows the small-angle X-ray diffraction (SAXD) and the wide-angle X-ray diffraction (WAXD) profiles of the PC70/HD bilayer aqueous solutions with various DPG concentrations. In the range of DPG = 0 to 30 wt %, where the samples were in a hydrogel state, the SAXD profiles showed a broad peak (Figure 5a), indicating the lack of interlamellar correlation as expected from the electron

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RESULTS DPG-Induced Disintegration of the Homogeneous Hydrogel in the PC70/HD Bilayer System. We visually investigated the effect of increasing the weight ratio of DPG on the dispersibility of the PC70/HD bilayer system. Figure 1 shows a photograph of the aqueous solutions of PC70/HD bilayers with various DPG concentrations. While the sample 6807

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Figure 2. Optical micrographs of the PC70/HD bilayers in the absence (a, b) and in the presence (c, d) of 40 wt % DPG. Micrographs (b) and (d) were taken under crossed nicols. No clear structure was seen in the absence of DPG (a, b) whereas a number of bright lines with strong anisotropy were seen in the presence of 40 wt % DPG (c, d). The lines seen under crossed nicols were brightest at 45° from the polarization axis of the polarizer. Bar indicates 20 μm.

Figure 3. Freeze-fracture electron micrographs of the PC70/HD bilayers in the absence (a) and in the presence (b) of 40 wt % DPG. The bilayer assumed a small sheet-like structure with an amorphous surface in the absence of DPG (a). Multilamellar assemblies composed of stacked flat bilayer sheets were observed in the presence of 40 wt % DPG (b). Bar indicates 1 μm.

microscopic observation (Figure 3a). On the other hand, the SAXD profiles of the sample solutions with 40 and 50 wt % DPG showed a very sharp peak at 0.16 nm−1 (Figure 5a). Together with the electron microscopic observation shown in Figure 3b, these results indicate that the addition of a high concentration of DPG induces the formation of highly ordered stacks of large flat bilayers with a lamellar spacing of 6.25 nm, squeezing out the interlamellar water to form a separate aqueous phase. Meanwhile, the WAXD profiles showed a single sharp peak at 2.43 nm−1 (0.412 nm) irrespective of DPG concentration (Figure 5b), suggesting that DPG molecules are predominantly partitioned into the aqueous phase and do not disturb the

highly ordered hexagonal packing of hydrocarbon chains. It is likely that the addition of DPG modifies the physicochemical properties of the aqueous phase to induce the multilamellar structure formation, leading to the disintegration of the homogeneous hydrogel. DPG-Induced Change in the Phase Transition Behavior. To gain insight into the interactions responsible for the DPG-induced disintegration of the hydrogel, we investigated the effect of increasing DPG on the thermotropic phase transition behavior by differential scanning calorimetry (DSC). Figure 6 shows the dependence of the DSC heating thermograms in the PC70/HD/DPG/water system on the weight concentration of DPG. In the absence of DPG, a sharp 6808

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Figure 6. DSC heating thermograms in the PC70/HD/DPG/water system. The lipid (PC70 + HD) concentration was 5 wt %. The chainmelting transition peak shifted constantly to lower temperature with increasing DPG concentration up to 50 wt % and leveled off with a further increase of DPG. The peak shape was kept nearly constant up to 30 wt % and broadened slightly above 40 wt %.

When the weight ratio of DPG and water was fixed at 1.0, the transition temperature was practically constant at 58.9 °C within experimental uncertainty irrespective of the lipid concentration (Figure 7). Moreover, the dependence of the

Figure 4. An anisotropic two-dimensional SAXD pattern of PC70/HD bilayers at DPG = 40 wt %. The reflection from multilamellar assemblies distributed in arcs with intensity maximum at 0.159 nm−1 (arrows), suggesting that the orientation of bilayer sheets was not fully at random in the range of the beam size.

Figure 7. Influence of the lipid (PC70 + HD) concentration on the chain-melting transition temperature. The weight fraction of DPG, WDPG = DPG/(DPG + water), was fixed to be 0.5 (DPG/water = 1:1). The chain-melting transition temperature showed no dependence on the lipid concentration as far as we measured.

Figure 5. SAXD (a) and WAXD (b) profiles of the PC70/HD bilayer aqueous solutions with various DPG concentrations. In the range of DPG = 0 to 30 wt %, the SAXD profiles showed a broad peak, indicating the lack of interlamellar correlation. The SAXD profiles of the sample solutions with 40 and 50 wt % DPG showed a very sharp peak at 0.16 nm−1, indicating formation of multilamellar structures. The WAXD profiles showed a single sharp peak at 2.43 nm−1 irrespective of DPG concentration, indicating the highly ordered hexagonal packing of hydrocarbon chains.

transition temperature on the DPG/water ratio was unaffected by the lipid concentration (Figure 8). In other words, the interaction between lipid and DPG molecules is not stoichiometric. Thus, the DPG/water ratio is the dominant determinant of the chain-melting phase transition temperature under the conditions that the weight ratio of PC70 and HD is suitable to form a stable hydrogel (PC70/HD = 3:2). These results supported the inference in the previous section that DPG affects the bilayer organization through modification of physicochemical properties of the aqueous phase but not through its direct interaction with lipid molecules.

endothermic peak corresponding to the chain-melting transition appeared at 70.5 °C. The chain-melting transition peak temperature decreased constantly with increasing DPG concentration up to 50 wt % and leveled off with the further increase of DPG while the peak shape was kept nearly constant up to 30 wt % and broadened slightly above 40 wt %. The breakpoint in the transition peak temperature dependence on the DPG concentration well coincided with the appearance of the multilamellar structures shown in Figures 3 and 5. In order to clarify the action of DPG on the PC70/HD bilayer system, we examined the dependence of the chainmelting transition temperature on the lipid (PC70 + HD) concentration while keeping the DPG/water ratio constant.

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DISCUSSION We found that the addition of a high concentration of DPG induces disintegration of the hydrogel composed of PC70 and HD (Figure 1). The freeze-fracture electron microscopic observations and the small-angle X-ray diffraction (SAXD) measurements revealed that the addition of DPG induced morphological change in PC70/HD bilayers from amorphous sheet-like structures to neatly stacked multilamellar structures 6809

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hydrophobic region of the bilayer to some extent in the DPPC/ alcohol system.32 Furthermore, the fact that the DPG/water ratio is the dominant determinant of the chain-melting phase transition temperature irrespective of the lipid concentration (Figures 7 and 8) indicates that there is no stoichiometric interaction between DPG and lipid molecules and DPG affects the lipid bilayer through modification of physicochemical properties of the aqueous phase. We infer that the DPGinduced decrease in the dielectric constant of the aqueous phase plays a key role in the disintegration of the hydrogel, because substitution of polyols with relatively high dielectric constants such as glycerol and sorbitol (relative dielectric constant ε = 46.53 and 35.5, respectively)34 for DPG (ε = 20.38)35 diminished the change of the main transition temperature and did not disintegrate the hydrogel (data not shown). Second, we discuss the effect of the dielectric constant of the aqueous phase on the interbilayer interaction according to the standard DLVO theory.36,37 In the absence of DPG, the hydrogel is formed as a result of the homogeneous distribution of sheet-like PC70/HD bilayers throughout the solution as described in the previous study.23,24 The homogeneous distribution is derived from the repulsive interaction between adjacent bilayers with a net negative charge due to acidic lipids in the PC70 mixture. We have proposed a new gelation mechanism; water continuum surrounding homogeneously distributed charged bilayer sheets with open edges works as the structural network in the PC70/HD/water gel. As the interbilayer repulsion based on the Gouy−Chapman electric double layer theory25 is strong enough compared to the van der Waals attractive force, every sheet-like bilayer may keep the distance from the adjacent bilayers as large as possible, leading to the homogeneous allocation of the interbilayer water. How does the DPG-induced decrease of dielectric constant of the aqueous phase modify these repulsive and attractive forces? In order to evaluate the effect of the dielectric constant,

Figure 8. Dependence of the chain-melting transition temperature on WDPG in the PC70/HD/DPG/water system. The lipid (PC70 + HD) concentration was varied from 2 to 15 wt % as shown in the figure. The transition temperature decreased depending merely on WDPG irrespective of the lipid concentration, suggesting indirect interaction between DPG and bilayer-forming molecules.

(here, we designate them as flat bilayer stacks; FBS) coinciding with a change in appearance of the hydrogel (Figures 3 and 5). Stacking of very large sheet-like bilayers was further supported by polarized optical micrographs and the appearance of anisotropic SAXD patterns (Figures 2 and 4). These results suggest that the disintegration of the hydrogel is caused by the formation of FBS accompanied by a decrease in the interbilayer distance (Figure 9). What is the formation mechanism of FBS? First, we discuss whether DPG molecules directly interact with the lipid bilayers or not. As the addition of DPG had almost no effect on the WAXD profile and the sharpness of the transition peak in the DSC thermogram, DPG must not disturb the lipid molecular packing in the bilayer. These results suggest that DPG with two hydroxyl groups is not incorporated into the hydrophobic region of the bilayer because it has higher hydrophilicity than ethanol, which is known to be able to be dissolved into the fluid

Figure 9. Schematic illustration of the morphological change of the PC70/HD/bilayer induced by the addition of DPG. The bilayer organization is transformed into unusually large flat bilayer stacks (FBS) by the addition of DPG as a result of the reduction of the aqueous medium dielectric constant ε. The FBS formation brings forth the disintegration of the hydrogel (see text for details). 6810

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(1997),39 the Hamaker constant is reduced by about 40% when 40 DPG with a refractive index n20 is added in equal D = 1.441 41,42 20 weight to water (nD = 1.333) (additivity of the refractive index and the dielectric constant is assumed). Thus, the addition of DPG may result in the decrease in the van der Waals attractive force via the decrease in the Hamaker constant. Shen et al. (2012)43 reported the increase in the interbilayer distance induced by the addition of glycerol and explained it by the refractive index matching between the bilayer and the solvent, which reduces the van der Waals attractive force (see eq 5). Since glycerol has a dielectric constant larger than DPG, it is likely that glycerol has an influence predominantly on the van der Waals interaction. According to the DLVO theory, the interaction energy per unit area W for the above model system is given by

let us consider a simplified model with two charged surfaces (surface charge density σ), between which an aqueous medium with a relative dielectric constant ε is filled in the absence of an electrolyte. The electric double layer theory gives the pressure between these two surfaces P as a function of the distance between them D.

⎛ kT ⎞2 P(D) = 2ε0εK 2⎜ ⎟ ⎝ e ⎠

(1)

where ε0 is the dielectric constant of a vacuum, k is the Boltzmann’s constant, T is the temperature, and e is the elementary electron charge. The value of K is calculated from the electric field at the surface Es.25 Es = (2kTK /e) tan(KD/2) = −σ /ε0ε

(2)

⎛ πkT ⎞2 1 A ⎟ W (D) = We(D) + Wν(D) ≈ 2ε0ε⎜ − ⎝ e ⎠ D 12πD2

If D is large, K is approximated to be π/D and the interaction energy We(D) can be easily calculated by integrating P(D) ∝ 1/ D2 (Langmuir equation).25 D

We(D) = −

∫∞

D

P(D) dD ≈ −

∫∞

(6)

⎛ π ⎞ ⎛ kT ⎞ 2ε0ε⎜ ⎟ ⎜ ⎟ dD ⎝D⎠ ⎝ e ⎠ 2

⎛ πkT ⎞ 1 ⎟ = 2ε0ε⎜ ⎝ e ⎠ D

2

The function W(D) has a local maximum at Dmax =

2

(3)

α=

This must be a good approximation for the hydrogel state of the PC70/HD system because the interbilayer water thickness is estimated to be very large (about 95 nm), assuming that the water molecules in the solution are equally allocated to every unit area on the bilayers, where the surface area per hydrocarbon chain is 0.196 nm2 derived from WAXD data (lattice spacing of 0.412 nm). The approximation of K by π/D is effective for the qualitative discussion on the FBS formation because the relative error in this approximation is not so large as to change the order of magnitude even with the D as small as ∼1 nm under our experimental conditions, i.e., ε = 20−80 and σ = 0.028 C/nm2 (estimated from the acidic lipid content in PC70). Additionally, the error gets smaller with decreasing ε of the aqueous phase by the addition of DPG. In fact, using the K value numerically calculated from eq 2 instead of π/D does not affect the qualitative conclusions below. According to the eq 3, We(D) decreases in proportion to the decrease of ε by the addition of DPG. For example, when DPG (ε = 20.38) is added in equal weight to water (ε = 80.10),34 ε and consequently We(D) are reduced by about 40%. On the other hand, the van der Waals energy between two flat surfaces per unit area is given by Wν(D) = −

A 12πD2

e2 [m /J ] 12ε0π 3(kT )2

(7)

and the maximum interaction energy is W (Dmax ) =

12ε0 2ε 2π 5(kT )4

Ae 12ε0 π (kT )4

4

=β×

ε2 , A

2 5

β=

e4

[m ·J ]

(8)

Since the interbilayer interaction energy W(D) of eq 6 has no secondary minimum, the sheet-like bilayers must separate as far as possible if the solvent content is high enough to make the thickness of the interbilayer solvent larger than Dmax. In this case, distribution of the interbilayer distance will be incoherent due to the lack of a local minimum in W(D). The hydrogel state is expected to be under these conditions. In fact, the SAXD profiles in the range of DPG = 0 to 30 wt % showed broad peaks (Figure 5a), indicating the lack of interlamellar correlation. When ε is decreased by the addition of DPG, Dmax is almost unchanged and W(Dmax) decreases roughly in proportion to ε (eqs 7 and 8) if the reduction rate of A is nearly equal to that of ε as discussed above. This means that the addition of DPG reduces the energy barrier for keeping the bilayers as far apart as possible and makes it easier to form the FBS. Furthermore, the reduction of ε may cause a decrease in the surface charge density of the PC70/HD bilayers because it makes the electric field near the bilayer surface stronger and decreases the local pH by enhancing accumulation of counterions (H+). As the reduction of surface charge density causes the decrease in W(Dmax) via eq 2, this effect may accelerate the decrease in the energy barrier. Although the addition of DPG certainly reduces the energy barrier, it is still very hard to overcome W(Dmax), which is the interaction energy per unit area on the order of 10−2 J/m2, with the help of the thermal energy being equally distributed to each mode kT because the bilayer sheets in our system are very large (>100 μm2). Hence, we speculate that the DPG-induced energy

(4)

where A is the Hamaker constant.38 The Hamaker constant depends on the material of the bilayer and the solvent properties. According to the Lifshitz theory, the Hamaker constant for our system can be described as follows:38 2 3hνe (nb2 − ns2)2 3 ⎛ εb − εs ⎞ A = kT ⎜ ⎟ + 4 ⎝ εb + εs ⎠ 16 2 (nb2 + ns2)3/2

Ae 2 A =α× , ε 12ε0επ 3(kT )2

(5)

where nb, εb, ns, and εs are the refractive indexes and relative dielectric constants of the bilayer and the solvent, respectively. The main electronic absorption frequency νe is typically around 3 × 1015 s−1. If we take the value of nb to be 1.56, which is roughly estimated from the table given by Huang and Levitt 6811

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force between neighboring charged headgroups, which could accumulate at the bilayer edge. This may result in an increase of the effective molecular surface area, which lowers the critical packing parameter of the acidic lipid molecule to reduce the stress for capping the bilayer edge. Thus, the unusually large flat bilayer in the FBS could be stabilized in the presence of DPG via the decrease in the dielectric constant. Finally, from the industrial point of view, it is very important to regulate solvent properties to stabilize the hydrogel state because modifiers of solvent such as polyol, which must be added to almost all cosmetic and pharmaceutical products for the purpose of moisturization or preservation,49−52 often causes serious damage to the hydrogel formation. In this context, the fact that the gelation, lamellarity, morphology, and the phase transition temperature of the hydrogel are able to be controlled by changing the dielectric constant of the aqueous medium is very useful information for the cosmetic and pharmaceutical industries to produce a unique, functional, and stable hydrogel. Furthermore, we demonstrated that PC70/HD bilayers are able to constitute a highly ordered FBS in the presence of DPG in spite of inhomogeneity of their lipid composition. Thus, a highly ordered assembly is attainable by simply modulating the dielectric constant of aqueous medium even in a crude system. These results offer a hint to understand the effect of solvent on the cosmetic and pharmaceutical formulations composed of multicomponent lipid systems.

barrier reduction makes it possible for the bilayer sheets to approach locally within the distance Dmax due to the partial bending of the membrane driven by thermal fluctuation. It should be noted that the undulation of the bilayer surface is clearly seen in the electron micrographs of the hydrogel (Figure 3a). If the attractive force exerted at the closely contacted portion is strong enough to overcome the repulsive force exerted in the surrounding portion, the bilayer sheets are able to form FBS. Since the magnitude of the attractive force depends on the steepness of the drop of W(D) at D < Dmax, we need to know the detailed landscape of the interaction energy around Dmax, which is on the order of 1 nm in our system. Here, we only point out that the effect of DPG on the hydration force must be considered as further theoretical analysis is beyond the scope of this experimental work. Now, we will discuss the behavior of SAXD profiles. The diffraction peak is very sharp in the presence of 40 and 50 wt % DPG whereas it is quite broad in the presence of less than 30 wt % DPG (Figure 5a). These results are consistent with the formation of FBS by the addition of a high concentration of DPG. However, the sharp peak from FBS structures appeared at a scattering angle larger than that for the maximum of the broad peak in the hydrogel state in spite of the fact that the thickness of the bilayer is smaller than the lamellar repeat distance, which contains the thickness of a water layer. A similar situation was reported in the dipalmitoylphosphatidylglycerol/ NaCl system, where the addition of NaCl induced the transformation from single- to multilamellar structures and the broad peak position (6.48 nm) in the absence of NaCl is at smaller angle than the sharp peak position (6.1 nm) in the presence of a high concentration of NaCl.44 These results can be explained by the simulation using simple electron density models.45 We checked whether the SAXD profiles in Figure 5a can be explained qualitatively based on the characteristics of the electron density profile of our sample by a simple simulation (see Supporting Information for details). The simulation suggested that the maximum position of the broad peak from single lamellar structures is at a smaller angle when the relative magnitude of the average electron density around the position of phosphorus ρp is smaller. These conditions must be satisfied in our system because PC70 is a mixture of various phospholipid species, and hexadecanol has no phosphorus. One of the conspicuous characteristics in our system is the exceptionally large size of the flat bilayer sheet (Figure 3). The morphology of PC70/HD bilayers was changed from small amorphous sheet-like structures to very large flat bilayer stacks (FBS) by the addition of a high concentration of DPG. Thus, DPG is likely to affect the size of the bilayer sheet, which could be regulated by the number of molecules stabilizing its open edge. Considering that the mixtures of HD and pure PC (or PC90) do not form the bilayer sheet,23,46 the edge-forming molecule must be a minor component of PC70 and have a small critical packing parameter proposed by Israelachivili47 to form a highly curved edge-capping. Acidic lipids with large effective headgroup areas such as phosphatidylglycerol (PG) may work as the edge-forming molecules, as the PG bilayer is known to assume a sheet-like structure.48 If it is the case, the addition of DPG may reduce the number of the edge-forming molecules to increase the size of a bilayer sheet because the DPG-induced decrease in the dielectric constant promotes protonation of the acidic lipids as already described in the discussion about the interbilayer interaction. In addition, the decrease in the dielectric constant may enhance the repulsive

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CONCLUSIONS

We investigated the effect of dielectric properties of solvents on the hydrogel composed of PC70 and HD by replacing water with water/DPG. We found that the addition of DPG caused disintegration of the hydrogel accompanied by morphological change in PC70/HD bilayers from mutually separated amorphous sheet-like structures to highly ordered flat bilayer stacks (FBS), the size of which was unusually large. Since the interbilayer distance in the FBS was much shorter than that in the hydrogel, the aqueous medium should be excluded from the interbilayer space to form a bulk aqueous phase. The effect of the solvent dielectric constant on the interbilayer interaction was semiquantitatively evaluated on the basis of the DLVO theory in the absence of an electrolyte. The calculation suggested that the DPG-induced decrease in the solvent dielectric constant could be a trigger for the morphological change via lowering the maximum interbilayer interaction energy W(Dmax) and/or the surface charge density. However, additional processes such as a local fluctuation of the bilayer surface are required to fully explain the mechanism of FBS formation because the decrease in the solvent dielectric constant cannot remove the energy barrier. The fact that simple modification of the solvent dielectric constant induced well-organized structural change in the system containing a crude lipid mixture PC70 suggests that even in a complex lipid mixture the behaviors of the bilayer assembly such as gelation, lamellarity, morphology, and phase are controllable by changing the solution environment. These findings will contribute to the further development of useful hydrogels consisting of plural amphiphilic molecules and the regulation of their physicochemical properties for industrial applications. 6812

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b01217. SAXD simulation result and the approximate phospholipid composition of PC70 (Table S1) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The synchrotron X-ray diffraction experiments were performed at BL40B2 of SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (Proposal Nos. 2014B1366, 2015A1190, and 2015A1406). This work was partially supported by the MEXT-Supported Program for the Strategic Research Foundation at Private Universities (S1201027) 2012-2016.

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REFERENCES

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