On the Molecular Interactions in Lipid BilayerâWater Assemblies of

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

On the Molecular Interactions in Lipid BilayerWater Assemblies of Different Curvature Martynas Talaikis, Maria Valldeperas, Ieva Matulaitiene, Jekaterina Latynis Borzova, Justas Barauskas, Gediminas Niaura, and Tommy Nylander J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b11387 • Publication Date (Web): 20 Feb 2019 Downloaded from http://pubs.acs.org on February 21, 2019

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The Journal of Physical Chemistry

On the Molecular Interactions in Lipid BilayerWater Assemblies of Different Curvature Martynas Talaikis †,a Maria Valldeperas ‖,a Ieva Matulaitienė ‡, Jekaterina Latynis Borzova †, Justas Barauskas #, Gediminas Niaura †,* and Tommy Nylander ‖,* † Department of Bioelectrochemistry and Biospectroscopy, Institute of Biochemistry, Life Sciences Center, Vilnius University, Sauletekio av. 7, LT-10257, Vilnius, Lithuania ‖ Department of Physical Chemistry, Lund University, P.O. Box 124, SE-22100 Lund, Sweden  NanoLund, Lund University, P.O. Box 118, SE-22100 Lund, Sweden. ‡ Department of Organic Chemistry, Center for Physical Sciences and Technology, Sauletekio av. 3, LT-10257, Vilnius, Lithuania # Camurus AB, Ideon Science Park, Gamma Building, Sölvegatan 41, SE-22379 Lund, Sweden. a

These authors contributed equally to the work

* Corresponding author: [email protected] [email protected]

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ABSTRACT

This work concerns the importance of the type of intermolecular interactions present in aqueous lipid-assembly systems depending on the type of aggregates they form. We have studied aqueous mixtures of diglycerolmonooleate (DGMO), Capmul glycerolmonooleate (GMO-50) and polyoxyethylene (20) sorbitanmonooleate (Polysorbate 80, P80) using small angle X-ray scattering measurements to reveal the structure of liquid crystalline phases. Based on SAXS data a phase diagram was constructed. We discuss the effect of curvature changes of the lipidaqueous interface obtained by changing the water content and the temperature. The results are related to the intermolecular interactions as revealed by Raman spectroscopy with focus on the bilayer type of system of different curvature and bilayer flexibility, namely lamellar, bicontinuous cubic phase and sponge phase. All phases show large similarities in their chain conformation and head group interactions as revealed by the Raman spectra, arising from the fact that all three structures are formed by lipid bilayers. However, subtle differences in molecular organization of the sponge phase were revealed by employing Raman difference spectroscopy and analysis of key spectroscopic indicators, which show less dense hydrocarbon chain packing compared to the inverse bicontinuous cubic or lamellar phase.


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INTRODUCTION Polar lipids are amphiphilic molecules that can self-assemble in presence of a solvent into a large variety of different structures, known as lipid liquid crystalline (LLC) phases. These structures have been widely studied not only due to their presence in living organism,1–3 but also for their tunable physicochemical properties both depending on the molecular structure and the environment, like temperature, solvent and solvent concentration. This is the reason why LLC have been used in multiple fields, as drug delivery systems,4–7 biosensors,8 for protein crystallization/immobilization9–12 or even as model systems to mimic biomembranes.13 LLC are formed to minimize the exposure of hydrophobic moieties of the lipid, i.e. the hydrocarbon chain, to water. Depending on the inter/intramolecular interactions and geometric packing of the amphiphiles used and on the environmental conditions (water content, temperature, pH, nature of electrolytes), different LLC will be formed.14–19 The packing parameter (Ns = VHC ahgl) is widely used to estimate the geometric packing properties of an amphiphilic molecule, as it correlates the acyl chain volume (VHC) with the product of the head group area (ahg) and chain length (l).14,15 When Ns of a lipid molecule is lower than 1, i.e. the cross-section area of the head group is larger than the acyl chain area, ‘oil-in-water’ structures are formed. On the other hand, when Ns is larger than 1, inverse structures (‘water-in-oil’) are formed. In each structure, the lipid film will bend towards the polar or apolar region. This is related to the concept of mean curvature (H) of a lipid film, which is positive for structures that curvature towards the polar region, and it is negative if it bends towards the apolar region.20 In addition, LLC phases with zero mean curvature can be found, like the lamellar (Lα) that it is arranged in planar bilayers or bicontinuous phases that are formed by lipid bilayer curved towards both polar and apolar regions.


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The inverse bicontinuous structures, such as cubic (Q) or sponge (L3) phases, have become of high interest due to their capability to entrap both hydrophobic compound in the bilayer and hydrophilic compounds in the aqueous pores. These structures form larger enough water pores, compared to other LLC, to be able to accommodate biological macromolecules such as proteins. While the different inverse bicontinuous cubic phases are well studied, the sponge phase structure is not as well understood. We have previously reported21 that at 25°C the quaternary DGMO/GMO-50/P80/water system can form highly swollen inverse bicontinuous cubic and sponge phases between the DGMO/GMO-50 ratios of 40/60 to 45/55. We concluded that the L3 phase is structurally related to the inverse bicontinuous cubic phase, as the peaks’ position of the two phases were very similar as observed by small angle X-ray scattering (SAXS) and also due to their neighboring location in the phase diagram. This suggested that the sponge phase represents a ‘melted’ and less ordered cubic phase, as previously proposed by Anderson et al.22 However, this is still not unambiguously established and further investigation was therefore required. Here we have therefore studied the phase behavior and structure along the dilution line at 40/60 DGMO/GMO-50 and 70/30 of total lipid/P80 (28/42/30 final wt % of DGMO/GMO50/P80) at different temperatures by SAXS and Raman spectroscopy. The aim is to provide new insight on the lipid sponge phase structure in the molecular level in relation to the bicontinuous cubic and lamellar phase. For this purpose we conducted studies on two different length scales. The first concerns the self-assembly level, where the SAXS analysis of the different LLC phases as a function of water content and temperature give us more insight into the phase behavior and the effect on the LLC curvature. The second concerns the molecular conformations and


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interactions, which were revealed by using Raman spectroscopy, which has proven to be a powerful tool to study lipid-aqueous liquid crystalline phases.23–26

MATERIALS AND METHODS Materials. A mixture of glycerides denoted as Capmul® GMO-50 (Lot No. 100616-8) was provided by Abitec (Janesville, WI). Its composition was 54.7% monoglycerides, 15−35% diglycerides and 2−10% triglycerides with the following fatty acid composition: 84.6 % oleic (C18:1), 6.8 % linoleic, 0.8 % linolenic and 6.2 % saturated acids. Diglycerolmonooleate (DGMO) with 88 % of diglycerol monoester and 4.9% free glycerol and polyglycerols was received from Danisco A/S (Brabrand, Denmark). The main fatty acid component was oleic acid constituting 90.7%, followed by linoleic (4.2 %), saturated (2.9 %), eicosenoic (1.2 %) and linolenic (0.8 %). Polyoxyethylene (20) sorbitanmonooleate (Polysorbate 80, P80) was supplied by Sigma-Aldrich and Croda (Chocques, France). Milli-Q purified water (18 MΩ·cm) and deuterated water (99.9% of D atoms, Sigma-Aldrich) were as well used. Sample Preparation. A stock lipid mixture of DGMO, GMO-50 and P80 was prepared by comelting at 40°C and further weighting of each compound (28/42/30 wt %) into a glass vials. The blend was mixed on a roller table for 24 h at room temperature, before the addition of appropriate amounts of water (from 10 to 60 wt %). The vials were sealed and centrifuged at 371x g after which the vials were turned up-side down and then again centrifugated. The centrifugation process with changing orientation of vials every second time was then repeated several times to ensure proper mixing. Finally, the samples were left to equilibrate at room temperature for at least 1 week before measurements. During this equilibration time samples were repeatedly centrifuged as described above in order to ensure homogeneity.


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Small Angle X-ray Scattering (SAXS). Bulk lipid liquid crystals (LLC) were measured using the SAXSLabGanesha 300XL instrument (SAXSLAB ApS, Skovlunde, Denmark). The instrument was equipped with a 2D 300 K Pilatus detector from Dectris and a Genix 3D X-ray source. The X-ray wavelength was 1.54 Å-1 and the data was collected in the q range of 0.012−0.76 Å−1 during 30 min. Samples were sealed at room temperature between two thin mica windows in a metallic block. All samples were analyzed at eight different temperatures (20 °C, 25 °C, 27.5 °C, 30 °C, 32.5 °C, 35 °C, 40 °C and 45 °C) with intervals of 20−30 min of equilibration time and regulated by an external recirculating water bath. The two-dimensional scattering pattern was radially averaged using SAXSGui software to obtain I(q). Raman measurements. Temperature dependent and polarization Raman experiments were conducted using inVia Raman microscope (Renishaw, UK) equipped with thermoelectrically cooled CCD camera and 1800 grooves/mm grating. The 532 nm laser excitation was restricted to 6 mW. Total integration time was set to 100 s. Raman spectra were collected using a long working distance 50× /0.50 NA objective lens (Leica). LinKam temperature control system PE95/T95 with temperature accuracy of 0.05 °C was used in temperature-controlled experiments. Measurements were recorded in a 1 mm thickness 1 ml quartz cell after 5 min equilibration time at each temperature. The wavenumber axes were calibrated with silicon standard according to the line at 520.7 cm-1. Raman measurements of liquid crystalline (LC) phases in H2O/D2O were performed on an Echelle

type

spectrometer

RamanFlex

400

(PerkinElmer,

Inc.)

equipped

with

thermoelectrochemically cooled to –50 oC CCD camera. Near-infrared 785 nm diode laser excitation and Raman signal were transmitted through a fiber optic cable. Laser power was set to 100 mW. The diameter of the laser spot was ~ 200 μm. Total integration time for each spectrum


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was 5000 s. The Raman frequencies were calibrated using the polystyrene standard (ASTM E 1840) spectrum. Intensities were corrected by a NIST intensity standard (SRM 2241). Frequencies, intensities, and FWHM values were determined by fitting the experimental contour with Gaussian-Lorentzian form components. Spectral analysis was performed by employing GRAMS/A1 8.0 (Thermo Scientific) software. The accuracy in determining the relative shifts of the well-defined Raman bands was less than ± 0.4 cm−1.

RESULTS AND DISCUSSION In this paper, we consider the evolution of self-assembly structure formed along the dilution line with 40/60 weight ratio of diglycerolmonooleate/Capmul glycerolmonooleate and with lipid/ Polysorbate 80 weight ratio of 70/30. We examine how temperature changes the self-assembly structure and relate these changes to the molecular interactions as revealed by Raman spectroscopy. The molecular structures of the main components in the system are shown in Figure 1. The spectroscopic regions of particular interest for a tail region of the lipid system are27,28 : (i) the C−C stretching modes (1000−1200 cm−1); (ii) the =C−H in-plane, deformation, in-phase methylene twisting, methylene bending, and C=C stretching modes (1260−1700 cm−1); and (iii) the C−H stretching modes (2800−3000 cm−1). In addition, several features that can be assigned to the head group region will be considered.


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Figure 1. Molecular structures of a) Diglycerolmonooleate (DGMO); b) Glycerolmonooleate (GMO50) and c) Polysorbate 80 (P80).

Phase behavior of the DGMO/GMO-50/P80 system. The study of structural changes as a function of temperature focus on the dilution line with 40/60 weight ratio of DGMO/GMO-50 and with lipid/P80 weight ratio of 70/30. The purpose of this choice is that this allows us to follow the evolution of a range of curvatures from lamellar to bicontinuous cubic phase, including the lipid sponge phase structure. The sponge phase is not as well characterized as other LLC phases and with this study we aim to increase our knowledge on the lipid molecular organization within the sponge phase. From our previous studies at 25 °C,21 we know that at low water content (10 wt%) a lamellar (Lα) phase is found. At a higher degree of hydration, the system forms inverse bicontinuous cubic phases with space group Ia3d (Q230, 20−40 wt%) and Pn3m (Q224, 50 wt%). Finally, above 60 wt% water, sponge (L3) phases are formed and coexist with excess water at larger hydrations.


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Here the effect of temperature on the structure of the lipid phases and hence the curvature of lipid-aqueous interface have been explored. SAXS have been used to determine the lipid liquid crystalline phase from 20 °C up to 45 °C at six different amount of added water (0−60 wt%). The water dilution series allows us to reveal the structural relationship between the L3 phase and the other LLC. We will focus our discussion on the three most relevant compositions (10, 40 and 60 % water), where we observed three “pure” LLC phases in water (H2O) and deuterated water (D2O). SAXS data on other compositions can be found in the Supplementary information (Figure S1). At 10 % hydration, lamellar (Lα) phase is found between 20 °C to 27.5 °C and inverse micellar (L2) phase above 40 °C. The former phase is characterized by the presence of two Bragg peaks with spacing ratio 1:2 (Figure 2a), while the L2 phase shows a single broad peak. It should be noted that at 30 oC the lamellar Bragg peaks are more pronounced, but wider as the system is close to the phase transition towards L2. At 40 % water, the inverse bicontinuous cubic phase a)

b)

c)

Figure 2. SAXS data of samples at 40/60 DGMO/GMO-50 with constant lipid/P80 ratio of 70/30 at different temperatures and hydrations of: a) 10%, transition from Lα to L2; b) 40%, transition from Q230 to H2; and c) 60%, L3. Temperatures are depicted on the graphs. Dashed line shows SAXS curves for samples in deuterated water instead of protonated water.


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with space group Ia3d (Q230) is predominant (Figure 2b). At least four Bragg peaks are identified at the relative positions of √6: √8: √14: √16, which corresponds to the Bragg reflections with Miller indices of (211), (220), (321) and (400). The phase transformation from a cubic phase into an inverse hexagonal (H2) phase above 35 °C is apparent from the presence of three Bragg peaks at the spacing ratios of 1: √3: √4. This is also supported by the calculated lattice parameter values (Figure S2a). As expected, when the temperature raises the lattice dimensions decreases as the curvature becomes more negative until it forms H2 phase. Interestingly, at the highest water content studied (60 %) the lipid inverse bicontinuous L3 (sponge) phase is formed at all the temperatures studied and no indication of a phase transition is detected (Figure 2c). However, it should be noted that the two structural peaks observed shift towards larger q-values with increasing temperature. This indicates that the repeat distances in the L3 phase and hence the water channels are reduced in size. Figure 2 (dashed line) shows the data from the samples prepared in D2O which form the same structures as the equivalent composition in water at 25 °C. It should be noted that the LLC prepared in D2O show a small shift toward higher q-values, as the sample was prepared by weight and D2O is denser than protonated water. No indication of the presence of P80 micelles was observed in the cubic and sponge phases based on an earlier thorough study of the phase diagram with and without P80. The corresponding diffractogram of the cubic and sponge phases looked similar with and without P80.21 Lattice parameter and water channel dimensions were estimated for these phases (Figure S2) as described in the Supplementary information.21,29–34 The values obtained for the inverse bicontinuous cubic and sponge phases agree quite well with the ones previously reported at 25 °C.21


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Based on the SAXS and visual inspection the phase diagram displayed in Figure 3 was constructed.

Figure 3. Phase diagram of samples analyzed by SAXS as a function of water content and temperature. Phases are indicated on the graph. Crosses depict multiphase region where: at 10 wt% water the sequence of phase transition is Lα to L2 transition; at 30 wt% it is Q230 to H2 transition; at 40 wt% it is mostly a mixture of Q230 and Q224 phases; and at 50 wt% is mostly Q224 at low temperatures. It was not possible to unambiguously determine the LLC structure at 50 wt% and high temperatures due to poor resolution of the diffractogram. The assignments of the Raman bands for the molecules within the self-assembled system. To insure the reliable assignments of the Raman bands of studied lipids we have recorded the polarized and low temperature Raman spectra of sponge phase. To separate symmetric and asymmetric vibrational modes the Raman spectra of parallel (𝐼 ∥ ) and perpendicular (𝐼 ⊥ ) polarizations with respect to the polarization of the incident laser beam were recorded and depolarization ratios (𝜌 = 𝐼 ⊥ /𝐼 ∥ ) were calculated. Raman bands with ρ ≥ 0.70 were considered as depolarized and the corresponding vibrations were therefore assigned to asymmetric modes. However, high frequency C−H bands were assigned to asymmetric modes based on


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characteristic frequency values and literature data.35–43 Raman bands possessing ρ ˂ 0.70 were considered as polarized. Observed bands with ρ lower than 0.20 were identified as obviously symmetric vibrational modes. Figure 4a shows the polarized Raman spectra for the sponge phase sample in the liquid (293 K) state. Here we note that we only consider the depolarization ratios for the sponge phase, since the samples with the other two phases appear slightly opaque as they contain larger amount of lipid (60 and 90 % in contrast to 40 % for sponge LLC). This makes the depolarization values less reliable. Figure 4b displays the low-temperature Raman spectrum of LLC. Under these conditions ordering of alkyl chains and intermolecular interactions of lipids are increased.43 Peak positions and depolarization ratios as well as assignments of the bands are based on previous studies of similar compounds24–26,35–43 and our own analysis of depolarization ratios and temperature-induced spectral changes. The results are listed in Table 1. Because of the low depolarization ratio, the bands at 849, 887, 1654, 1740, 2848, and 2867 cm−1 are assigned to symmetric vibrational modes, while depolarized bands associated with asymmetric vibrational modes were find at 970, 1299, 1437, 1453, and 1472 cm−1.

Table 1. Positions (ν), depolarization ratios (ρ) and assignments of the Raman vibrational modes of the sponge lipid liquid-crystalline (LLC) phase. Solid K) ν (cm−1)

(223

Liquid (293 K) ν (cm−1)

ρ

730 vw

725 vw

0.40

791 vw

810 vw

0.20

νs(C–O), δ(CCO)

852 w

849 m

0.15

νs(C–O−C)

Assignments

693 vw


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873 vw

0.24

888 m

887 w

0.15

922 vw

912 vw

0.20

940 vw

941 vw

971 vw

970 vw

0.74

1015 vw

0.11

1047 vw

1042 w

0.26

νs(C–O–C)

1064 m

1063 w

0.40

ν(C–C)T

1095 m

1085 m

0.56

ν(C–C)T

1121 m

1119 w

0.34

ν(C–C)T, νas(C–O–C)

1135 vw

1140 vw

0.47

1248 vw

1239 vw

0.53

1272 m

1262 m

0.59

1282 w

0.69

1295 s

1299 m

0.74

1309 vw

1315 w

0.63

1346 w

0.61

1365 w

0.34

1438 s

1437 m

0.71

δ(CH2) scissoring

1450 w

1453 m

0.75

δ(CH2) scissoring

1467 m

1472 w

0.76

δ(CH3) scissoring

1658 m

1654 s

0.16

ν(C=C) cis conformation

1716 vw

1723 vw

0.19

ν(C=O)

1733 vw

1740 w

0.14

ν(C=O)

2843 s

2848 s

0.10

νs(CH2)

2862 s

2867 s

0.10

νs(CH3)

r(CH3)T

δ(CH2)

982 vw

δ(=C–H) cis conformation

t(CH2)


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2881 s

2888 s

0.27

νas(CH2)

2900 s

2907 s

0.35

νs(CH2)FR

2928 s

2930 s

0.18

νs(CH3)FR

2958 s

2957 s

0.30

νas(CH3)

3002 m

3004 m

0.20

ν(=C–H)

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Abbreviations: ν – stretching, νs – symmetric stretching, νas – asymmetric stretching, δ – deformation, δas – asymmetric deformation, τ – twisting, r – rocking, wag – wagging, T – trans, G – gauche, n.d. – not detected, sh – shoulder, w – weak, m – middle, s – strong, vs – very strong, FR – Fermi resonance.

Figure. 4. Polarized Raman spectra of sponge LLC phase sample at room temperature (a), and spectra for the same sample at 223 K (b) in the middle and high frequency spectral range. Water content in the sample was 60 wt %. Excitation wavelength is 532 nm, total integration time 500 s. The molecular conformation and interactions of lipids in self-assembly structures as revealed by Raman Spectroscopy. Water content, lipid composition and temperature are key elements in determining the packing parameter of the lipids and hence the formation of particular liquid crystal phase. The overall spectral intensity in Figure 5−8 decreases with a factor of about


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1.3 when comparing the spectra of lamellar and cubic phases and of about 2.1 when comparing lamellar and sponge phase spectra. This is consistent with different lipid content in the sponge, cubic, and lamellar phases that contain 40, 60, and 90 wt % of lipid+P80, respectively. Overall the changes in the spectra between different samples are rather small, which confirm that they are all composed of lipid bilayers although the curvature is different. In order to pin-down minor differences that occur between key spectral markers of LLC phase, the difference spectra were calculated. These were obtained by normalizing the intensity of the spectra based on the dominant vibrational mode of the stretching C=C bond near 1655 cm−1. Figures 5−8 show Raman spectra of selected regions of sponge (L3), cubic (Q230) and lamellar (Lα) LLC phases in H2O/D2O. The analysis of the data will be following the regions of interest for lipids as previously mentioned: (i) 680−1170 cm−1; (ii) 1180−1550 cm−1; (iii) 1600−1800 cm−1; and (iv) 2770−3050 cm−1. Spectra recorded in both H2O and D2O are shown to get further information on the head group solvent interaction. The difference spectra are also included in the figures with the purpose of highlighting the features that can be attributed to transition between the phases.


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Figure 5. Raman spectra of sponge (a), cubic (b) and lamellar (c) LLC phases in H2O (upper panel) / D2O (bottom panel) in spectral region of 680−1170 cm−1. Difference spectra of sponge-lamellar (d), cubic-lamellar (e), and sponge-cubic (f) are also shown. The spectra were normalized according the intensity of a peak near 1655 cm−1. Excitation wavelength is 785 nm. The intensity of difference spectra is multiplied by 10. (i) Tail and glycerol chain conformation (680−1170 cm−1): This region mainly covers the COC and CC stretching and CH2/CH3 rocking vibrational regions of LLC phases as shown in Figure 5. Thus it gives information on both the acyl chain and on the interaction in the glycerol head group. Here we note that apart from the GMO-50 and DGMO, the COC stretching signal from the P80 should also be considered. The most intense positive-going broad feature in the difference spectra near 850 cm-1 is the polarized band assigned to νs(C–O−C) vibrational mode. This band shifts to higher wavenumbers with increasing amount of water within the sample (cf. Figure 5 d and e). Similar blue shift was observed for this band in the spectrum recorded at lower temperature (Figure 4). Thus, spectroscopic changes in the νs(C–O−C) vibrational mode indicate an increase in hydrogen bonding interaction strength at C–O−C segment of studied lipid aqueous phases, when going from Lα to L3 structure. Similar tendency was observed in the spectra acquired from lipid assemblies in heavy water. It should also be noted that sponge and cubic phase spectra are generally very similar. However, the comparison of L3 and Q230 difference spectrum (Figure 5f) shows low intensity positive-going feature near 853 cm−1, indicating increase in hydrogen bonding interaction strength in the vicinity of head group of lipids for L3 phase comparing with Q230 one. In addition, the low intensity feature due to COC stretching vibration appears at 784 cm−1; this band is absent


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in the spectra of lipids assemblies prepared with heavy water, again suggesting association of this mode with vibration of COH group. Difference spectra also reveal changes in C−C stretching frequency region; the negative-going feature at 1081 cm−1 indicates decrease in population of C−C bonds in trans conformation going from Lα to L3 phase (Figure 5d). Relative intensity of a ν(C−C)T band could serve as intermolecular order of acyl chain indicator. Increased fluidity of hydrocarbon chains generates gauche defects and therefore the intensity of a ν(C-C)T band decreases. Thus transition to L3 or Q230 phases from Lα structure results in loss of all-trans chain segments. The same holds for heavy water samples. We can now rationalize this in terms of the critical packing parameter (𝑁𝑠 = 𝑉𝐻𝐶/(𝑙 ∙ 𝑎ℎ𝑔)), which is defined as the ratio between the volume of the hydrophobic chain (VHC) and the product of the head group area (ahg) and the chain length (l).14,15 Less fluid chains, i.e. more trans conformation, will decrease the packing parameter and hence favor the Lα phase over the L3 or Q230 phases. (ii) Conformation around the double bond and in the acyl chain region (1180 – 1550 cm−1): The most significant difference between sponge and cubic phase compared to lamellar phase in the spectral region from 1180 to 1550 cm−1 appears in the vicinity of 1272 cm−1, which in the difference spectra appears as a negative peak (Figure 6). This feature is associated to extremely conformation sensitive in-plane bending vibration of olefinic hydrogen atom [δ(=CH)ip], i.e. close to the double bond in the oleic acid chain. This frequency corresponds to cis form structure37 and it is a sharp and well-defined band in low temperature (solid state) spectrum (Figure 4). Thus, transition from Lα structure to L3 and Q230 phases results in loss of the solid state-like clusters in the vicinity of olefinic group. This could be attributed to the fact that the packing of the amphiphilic molecules present is less efficient for inverse bicontinuous phases (L3


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and Q230), i.e. larger volume of the chain region, than for the lamellar phase, which also is less hydrated.


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Figure 6. Raman spectra of sponge (a), cubic (b) and lamellar (c) LLC phase samples in H2O (upper panel) / D2O (bottom panel) in spectral region of 1180−1550 cm−1. Difference spectra of sponge-lamellar (d), cubic-lamellar (e), and sponge-cubic (f) are also shown. The spectra were normalized according the intensity of a peak near 1655 cm−1. Excitation wavelength is 785 nm. The intensity of difference spectra is multiplied by 10. In the region of CH2 scissoring vibrations a negative peak is visible in the differential spectra near 1441−1431 cm−1. Subtle changes in the interaction between the methylene groups are found in this region. There are not only differences in molecular interactions between L3 or Q230 and the Lα phase, that appear to have a higher intensity peak than the other two phases, but also between the more ordered (Q230) and disordered (L3) bicontinuous structures, as the cubic phase shows a higher intensity peak. This is apparent in Figure 6f where the comparison of L3 and Q230 phases also reveals a clear dip in the differential spectrum near 1431 cm−1. These results may arise from a larger molecular packing efficiency (or smaller acyl-chain volume) of the acyl chain present in the lamellar phase than in the inverse bicontinuous cubic phase, which becomes even less densely packed in the sponge phase. Similarly, it can be noted that the solid state spectrum of L3 phase (Figure 4b) shows considerable increase in relative intensity of δ(CH2) mode near 1438 cm−1. (iii) Interactions in the head group region (1600 – 1800 cm−1): Raman band of carbonyl group ν(C=O) is a composite of two components found at 1724 and 1742 cm−1 (Figure 7). These bands arise from a different degree of hydration of the C=O group.38 The band at lower wavenumbers is involved in hydrogen bonding with water, while the other belongs to unbound group. The relative intensity I1724/I1742 correlates with the amount of water within the samples. The proportion shifts from 0.8 to 1.5 for lamellar to sponge phase transition in water and from


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1.0 to 2.4 for LLC phases in heavy water (Table 2). These results could be related to the structures formed at higher water content. As the water amount increases, the lipids selfassemble in more curved structures in order to minimize the contact of the acyl chain (apolar) with water. In this case, this leads to the formation of inverse bicontinuous structures which have a larger surface area to volume ratio compared to the lamellar phase. Therefore, the contact with water is larger and thus, more hydrogen bonds with water are formed. Table 2. Intensity ratio between components of ν(C=O) band, I1724/I1742, of lamellar, cubic and sponge lipid liquid crystal phases prepared in water and heavy water. I1724/I1742

I1724/I1742

(H2O)

(D2O)

Lamellar

0.8

1.0

Cubic

1.3

2.0

Sponge

1.5

2.4


21


Figure 7. Raman spectra of sponge, cubic and lamellar LLC phases in H2O (left side panel) / D2O (right side panel) in stretching C=O region. Gaussian-Lorentzian shape components indicating free and bonded carbonyl groups are also included. Excitation wavelength is 785 nm. (iv) The order of the lipid matrix and the interaction between the chains (2770 – 3050 cm−1): The order of the lipid matrix and the interaction between the chains could be deduced by examining C−H stretching vibrations of the alkyl chains in the high frequency region of the spectra (Figure 8). The most distinctive features in the difference spectra are: the dip near 2869 cm−1 associated mostly with symmetric stretching vibrations of methyl groups; and positive-going band near centered at 2963 cm−1 associated mainly with asymmetric stretching vibration of methyl groups. These effects are more pronounced in the D2O than in the H2O spectra. This suggests that water induces perturbations in the vicinity of the terminal CH3 residues going from sponge and cubic phases to lamellar structure. This could be related to the different packing space given to these residues in the different LLC structure. While in the Lα the lipids and P80 molecules are arranged in a non-curved stack of layers, curved bilayers are found in the L3 and Q230 phase. These curved regions require a molecular packing different than the planar ones which might expose the terminal CH3 residues to water. This could be supported by the observations made by Mihailescu et al.,39 where they reported that more than 20 % of acyl chain methyl groups where in close contact with water in the liquid-disordered DOPC bilayer interface. In the frequency range of ν(=C–H) mode, a low intensity positive-going feature is visible near 3014−3016 cm−1 indicating an upshift in wavenumbers of the olefinic group =C–H stretching mode for L3 phase comparing with Lα structure. Figure 4b shows that lowering the temperature


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results in a downshift of the frequency in this mode. Thus, the observed trend is consistent with disappearance of clusters of more ordered structures near olefinic group in the sponge phase. Such effect is less expressed comparing the Q230 and Lα phases.


23


Figure 8. Raman spectra of sponge (a), cubic (b) and lamellar (c) LLC phase samples in H2O (upper panel) / D2O (bottom panel) in spectral region of 2770−3050 cm−1. Difference spectra of sponge-lamellar (d), cubic-lamellar (e), and sponge-cubic (f) are also shown. The spectra were normalized according the intensity of a peak near 1655 cm-1. Excitation wavelength is 785 nm. The intensity of difference spectra is multiplied by 10. Temperature effect on the acyl chain conformations and molecular interactions. Raman spectroscopy was utilized to determine conformational changes of the lipid m atrix in the temperature range between 20 and 45 °C. Figure 9 shows the spectra of LLC phase containing 10 wt% of water at 45 and 20 °C with temperature-difference spectra (45 °C minus 20 °C) of phases containing 10, 40, and 60 wt% of water. A set of difference spectra is presented in Figures S3−S5. The spectrum recorded at 20 °C was chosen to be a reference spectrum. The C=C stretching band at 1654 cm−1 and the high frequency =C−H stretching vibration at 3005 cm−1 were chosen to normalize the spectra intensity in the fingerprint (Figure 9a) and C−H stretching (Figure 9b) vibration regions. The difference between the three compositions studied will be analyzed by looking at the most important features in the difference spectra and at six different spectroscopic structure indicators depicted in Table 3. The observed spectral intensity diminishes as water content in samples increases and the spectral perturbations emerge only at higher temperature values. It should be noted that the spectral changes are completely reversible as difference spectrum obtained at 20 °C after temperature-dependent experiments shows no spectral features. Figures 9, S3−S5 depict the spectra of each composition as a function of temperature. As it can be noticed, there are common features observed on the three compositions as temperature is increased. The difference spectrum in the fingerprint area (Figure 9a) of all samples exhibits two


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negative going features around 1294 and 1432 cm−1 assigned to twisting and scissoring deformations of methylene group, respectively. In addition, low intensity dips are visible at 1063 and 1117 cm−1, corresponding to C−C stretching vibrations. The bands located at 1064, 1121, and 1295 cm−1 are markers of alkyl chain all-trans conformation (Table 1). Hence, the decrease in intensity of these bands indicates progressive loss of all-trans conformation with increasing temperature, most likely associated with the increase in chain motion. Similarly, in the high frequency region several negative-going features are assigned to symmetric and asymmetric CH2 stretching vibrations found at 2847 and 2885 cm−1; these bands are intense in the solid state spectrum of sponge phase recorded at 223 K (Figure 4b). Concurrently, the peak position of νs(CH2) band shifts to higher wavenumbers with increasing temperature. This trend is clearly visible in Table 3a. These results indicate that temperature induces intermolecular motion of hydrocarbon chain and decoupling of the interaction between chains.26,40 The perturbation in difference spectra appear gradually with almost linear intensity gain indicating consistent but steady transformations of conformational order of the molecules in the chosen temperature region. These features at low and high frequency regions could be related to the packing parameter (Ns = VHC/(l ∙ ahg)) of the lipids and P80 used here. It is well known that Ns increases with temperature, in particular because of the increase in acyl chain mobility and hence an apparent increase of the VHC. In addition, the head group effective size is reduced due to lower interactions with water. The increase in the packing parameter will affect the arrangement of these molecules to a more negative spontaneous curvature structure, like the L2 phase in the case of 10 % water or H2 phase in the case of 40 % water. Therefore, the acyl chain becomes more disordered by forming more gauche conformation (at the expense of the trans), which is a sign of more mobile


25


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hydrocarbon chains. Accordingly, this will allow the loss of inter-chain interactions in order to form more favorable LLC structures. In the case of the sponge phase (60 % water), no phase transition was observed in the temperature range studied. However, from the SAXS data was deduced that the unit cell dimensions for this phase decreases with increasing temperature, i.e. the system can uptake less water, which it is also a sign of a change towards a more negative curvature. Therefore, the same conclusions can be applied for this composition. Analysis of νs(CH2) dependence on temperature (Table 3a) shows transition point near 28 °C for Q phase, while linear increase in frequency with temperature was observed for both Lα and L3 phases. Importantly, the slope of frequency change increases in the order 0.027 > 0.040 > 0.053 cm−1/°C for lamellar, cubic, and sponge phases, respectively. Because νs(CH2) frequency is sensitive indicator of coupling between the alkane chains,40 observed different slopes point on the progressive increase in mobility of chains with increasing water content. Table 3 Comparison of various spectral indicators in temperature range of 22.5 and 45 °C of liquid crystal phases containing 10, 40 and 60 % of water. Raman indicator

10 %

a) νsCH2 (2848 cm−1) Alkyl chain coupling indicator; decoupling results in frequency upshift

b) I(νasCH2)/I(νsCH2) (2880 / 2848 cm−1) Rotational and conformational order indicator; ratio decreases with increasing disorder


26

40 %

60 %

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c) I(νsCH3FR)/I(νsCH2) (2930 / 2848 cm−1) Intermolecular interactions of alkane chains indicator; highly sensitive to coupling interactions affecting the terminal methyl group; decrease of intermolecular interactions (decoupling) results in increase of ratio intensity

d) tCH2 (1300 cm−1) Degree of coupling between alkane chains indicator; decoupling results in frequency upshift e) tCH2 FWHM (cm−1) Rotational disorder and gauche/trans conformation indicator; FWHM increases with increasing rotational disorder and number of gauche conformers in alkane chains f) I(CCG)/I(CCT) (1085 / 1120 cm−1) Gauche/Trans conformation of alkane chain indicator


27


To obtain further insight on the order/disorder state of the alkene chain as a result of interchain interactions, the intensity ratio between asymmetric and symmetric CH2 stretching vibrations [I(νasCH2/I(νsCH2)] was estimated (Table 3b). As a general trend, it can be observed that the ratio decreases with increasing temperature. This is associated to the decrease in the chain-chain vibrational coupling and an increment in the acyl chain mobility, also observed as an increase in gauche conformations.26,40 It was previously suggested23 that the I2885/I2850 ratio decreases going from Lα to H2 phase or from Q to L2 phase, i.e. it decreases when the lateral packing of disordered hydrocarbon chains is less constrained. This agrees quite well with the [I(νasCH2/I(νsCH2)] values at higher temperatures where the ratio increments from H2 < L2 ≤ L3, as the sponge phase is more hydrated and more flexible bilayer compared to other bicontinuous phases. However, if we consider the ratio at lower temperatures, the values increment from Q < Lα < L3. These differences could arise from the quaternary system used here, compared to the binary systems studied by Larsson et al.23 Therefore, indicator (b) in Table 3 might not be only dependent on the lateral packing density of one single type of acyl chains, but due to the complexity in terms of lipid composition considered. What is evident from this indicator is that the lipid L3 phase is more hydrated and possesses much disordered and loose hydrocarbon chain compared to other LLC phases. The band attributed to Fermi resonance of CH3 symmetric stretch appears at 2940−2942 cm−1 as positive feature in difference spectrum, while the methyl symmetric stretch found as negativegoing feature near 2847 cm−1. Intensity of CH3 FR band increases with increasing decoupling of alkane chains in the vicinity of terminal methyl group. Such interactions might be sensitively probed by using indicator I(νsCH3FR)/I(νsCH2)7,40 presented in Table 3c. It can be noticed that this marker is quite constant through all the temperature range studied. However, the values


28

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differ depending on the composition. In the case of low water content (Lα/L2 phase) the intensity ratio is around 0.44 indicating high coupling between the alkane chains in the vicinity of methyl groups. Addition of water to 40 % (Q/H2 phase) results in considerable ratio increase (0.75) indicating that water induces a decrease in interactions between the chains. This suggests that there is larger inter-chain separation/decoupling and that the methyl group has a larger rotational disorder at 40 % compared to 10 % water.40 Surprisingly, further increase in water content shows decrease of indicator value. This indicates an increase in ordering in the vicinity of methyl groups for L3 phase. The reason for this is not clear, but can possibly be assigned to the less curved structure of the more swollen L3 phase, which might restrict the movement of the methyl groups. The prominent band around 1300 cm−1 corresponding to the twisting vibration of methylene groups (tCH2) provides useful information on degree of coupling between the chains (peak position of the band) and rotational disorder and gauche/trans conformation (full width at half maximum, FWHM of the band).40 As one can see from the Table 3d and e, the dependence of tCH2 frequency on temperature differs considerably for 10, 40 and 60 wt% water samples. At 10 % water the values are lower compared to other formulations suggesting that the Lα/L2 phase at this composition present less inter-chain decoupling and lower intramolecular motions. In addition, other noticeable differences in each marker could be related to the LLC structures present in the sample. The most interesting one is at 40 % water. It presents a sudden frequency peak upshift at 32.5 °C which coincides with a phase transition from inverse bicontinuous cubic to inverse hexagonal phase. This increase in frequency could be related to an increase in “freedom” of the acyl chains movements present in H2 structure. Another noteworthy characteristic is seen at 10 %, where the FWHM is almost constant when there is only Lα phase


29


(until 30 °C) and raises when the phase transitions towards L2 phase starts. Other changes in tCH2 wavenumber and FWHM could be related to the swelling/shrinking of the sample (for example in the L3 phase) or to the GMO-50 melting around 40 °C. The peak high intensity of the band at 1085 cm−1 relative to the band at 1120 cm−1 is evaluated in indicator I(CCG)/I(CCT) (Table 3f). These peaks are related to the alkyl chain in gauche and trans conformation, respectively. As a general characteristic, it could be seen that the C−C gauche conformation is more predominant than the trans, as the ratio values are larger than 1. It is quite difficult to distinguish other features, as there is no general trend in any of the formulations and there is no much change with temperature. However, it can be seen that the ratio value for sponge phase is larger than for lamellar phase. This suggests that the acyl chains in the L3 phase are more disordered than in the Lα phase.26,40

Figure 9. Raman spectra of LLC phase containing 10 wt.% of water (upper side) at 45 °C and 20 °C as well as difference spectra of phases containing 10, 40 and 60 wt.% of water (bottom side) in fingerprint (a) and CH stretching vibration (b) region. Excitation wavelength is 532 nm.


30

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CONCLUSIONS Structure, phase behavior, and molecular interactions within lipid self-assembly structures with different curvature have been studied by small angle X-ray scattering and Raman spectroscopy. This work was performed to gain further knowledge on the lipidic sponge phase, as its structure is not as well-known as other lipid liquid crystal phases. The DGMO/GMO-50/P80/water system (dilution line 40/60 DGMO/GMO-50 ratio and lipid/P80 70/30) was analyzed as function of water content and temperature to fulfill this purpose. Raman evaluation indicates that the lamellar (Lα), cubic (Q230) and sponge (L3) phases present very similar features, probably arising from the fact that all three structures are formed by lipid bilayers. However, the difference spectra that points key spectroscopic indicators, which shows subtle but important differences, where the sponge phase shows less dense hydrocarbon chain packing compared to the inverse bicontinuous cubic or lamellar phase. Our study has demonstrated that the Lα phase present less fluid hydrocarbon chains, which can be interpreted as more solid-like clusters in the vicinity of the double bond compared to the inverse bicontinuous phases studied (Q230 and L3). In addition, differences in the CH2 scissoring vibrations were found as function of the molecular packing of the acyl chain, including differences between the Q230 and L3 phase which confirms that the sponge phase has a looser hydrocarbon chain packing compared to the inverse bicontinuous cubic or lamellar phase. Increasing the temperature induces an increase in the acyl chain motion (increase in gauche conformations) and decoupling between chains. This turns into a larger apparent volume of the hydrocarbon chain and therefore, an increase in the packing parameter (Ns) of the lipid/P80 molecules. Consequently, this favors the formation of structures with larger negative curvature as


31


supported by SAXS analysis. Moreover, it was found that the L3 phase presented a more disordered acyl chain compared to other phases. All of these results support the fact that the lipid sponge phase has a close structural relation to the lamellar and inverse bicontinuous cubic phases. However, it is a much more flexible bilayer which can adapt better to large uptakes of water and therefore, form larger unit cell dimensions.

ASSOCIATED CONTENT Supporting Information. SAXS data as function of temperature for samples with 0, 20, 30 and 50 wt% water; Lattice parameter and water channel diameter as a function of water content and temperature for different LLC; Temperature-difference Raman spectra of samples at 10, 40 and 60 wt% water. ACKNOWLEDGMENT This work was financially supported by the BIBAFOODs project from the European Union's Seventh Framework Programme FP7/2007-2013 as well as by a grant from Swedish Research Council (2016-05390). ABBREVIATIONS P80, Polysorbate 80; DGMO, diglycerol monooleate; GMO-50, Capmul® GMO-50; SAXS, Small Angle X-ray Scattering; LLC, lipid liquid crystalline; Lα or LAM, lamellar phase; L3 or SPO sponge phase; Q230 or CUB, cubic phase with Ia3d space group; H2, reverse hexagonal phase; L2, reverse micellar phase. REFERENCES


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Bryant, M. A.; Pemberton, J. E. Surface Raman Scattering of Self-Assembled Monolayers Formed from 1-Alkanethiols: Behavior of Films at Gold and Comparison to Films at Silver. J. Am. Chem. Soc. 1991, 113 (22), 8284–8293.

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Ho, M.; Pemberton, J. E. Alkyl Chain Conformation of Octadecylsilane Stationary Phases by Raman Spectroscopy. 1. Temperature Dependence. Anal. Chem. 1998, 70 (23), 4915– 4920.


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TOC GRAPHIC


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Page 38 of 48

OH

OH

Page 39 of 48 (a)

HO

1 2 3 HO (b)4 5 6 7 8HO CH2CH2O w 9 10 (c) O 11 12 w + x + y + z = 20 13 14


O

O

O OH O O OCH2CH2 O H x OCH2CH2 O H y OCH2CH2ACSOParagon z O

Plus Environment

a)

10 %

b)

40 %

60 %

c)

The Journal of Physical Chemistry Page 40 of 48 1 2

32.5oC

45 C 40oC

35oC

45oC 40oC

32.5oC

log I (a.u.)

40oC

35oC

o

log I (a.u.)

log I (a.u.)

45oC

35oC 32.5oC

ACS Paragon Plus Environment 30oC

27.5oC 25oC

30oC

27.5oC 25oC

20oC

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

q (nm-1)

30oC 27.5oC 25oC

20oC

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

q (nm-1)

20oC

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

q (nm-1)

Temperature (oC)

H2 Chemistry Page The 41 ofJournal 48 of Physical 45

40

1 35 2 3 30 4 5 25 6 7 20 8 9 0 10

L2 L3

Lα

Q224

Q230

ACS Paragon Plus Environment 5

10

15

20

25

30

35

40

Water (wt%)

45

50

55

60

65


Figure. 4. Polarized Raman spectra of sponge LLC phase sample at room temperature (a), and spectra for the same sample at 223 K (b) in the middle and high frequency spectral range. Water content in the sample was 60 wt %. Excitation wavelength is 532 nm, total integration time 500 s. 82x81mm (600 x 600 DPI)


Page 42 of 48

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Figure 5. Raman spectra of sponge (a), cubic (b) and lamellar (c) LLC phases in H2O (upper panel) / D2O (bottom panel) in spectral region of 680−1170 cm−1. Difference spectra of sponge-lamellar (d), cubiclamellar (e), and sponge-cubic (f) are also shown. The spectra were normalized according the intensity of a peak near 1655 cm−1. Excitation wavelength is 785 nm. The intensity of difference spectra is multiplied by 10. 82x185mm (600 x 600 DPI)



Figure 6. Raman spectra of sponge (a), cubic (b) and lamellar (c) LLC phase samples in H2O (upper panel) / D2O (bottom panel) in spectral region of 1180-1550 cm-1. Difference spectra of sponge-lamellar (d), cubiclamellar (e), and sponge-cubic (f) are also shown. The spectra were normalized according the intensity of a peak near 1655 cm-1. Excitation wavelength is 785 nm. The intensity of difference spectra is multiplied by 10. 82x172mm (600 x 600 DPI)


Page 44 of 48

Page 45 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7. Raman spectra of sponge, cubic and lamellar LLC phases in H2O (left side panel) / D2O (right side panel) in stretching C=O region. Gaussian-Lorentzian shape components indicating free and bonded carbonyl groups are also included. Excitation wavelength is 785 nm. 82x66mm (600 x 600 DPI)



Figure 8. Raman spectra of sponge (a), cubic (b) and lamellar (c) LLC phase samples in H2O (upper panel) / D2O (bottom panel) in spectral region of 2770−3050 cm-1. Difference spectra of sponge-lamellar (d), cubiclamellar (e), and sponge-cubic (f) are also shown. The spectra were normalized according the intensity of a peak near 1655 cm-1. Excitation wavelength is 785 nm. The intensity of difference spectra is multiplied by 10. 82x189mm (600 x 600 DPI)


Page 46 of 48

Page 47 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 9. Raman spectra of LLC phase containing 10 wt.% of water (upper side) at 45 °C and 20 °C as well as difference spectra of phases containing 10, 40 and 60 wt.% of water (bottom side) in fingerprint (a) and CH stretching vibration (b) region. Excitation wavelength is 532 nm. Lamellar phase spectra at 45 °C and 20 °C (upper part) and temperature-difference spectra of Lamellar, Cubic and Sponge phases (bottom part) in fingerprint (a) and CH stretching vibration (b) region. Excitation wavelength is 532 nm. 82x76mm (600 x 600 DPI)


Inverse micelles

The Journal of Physical Chemistry Page 48 of 48 Sponge

Inverse Cubic Ia3d

Temperature

1 2 Lamellar 3 4 5 6

Inverse hexagonal

Ns

ACS Paragon Plus Environment Water

vs

On the Molecular Interactions in Lipid BilayerâWater Assemblies of

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