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Rheology of ultra-swollen bicontinuos lipidic cubic phases Chiara Speziale, Reza Ghanbari, and Raffaele Mezzenga Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00737 • Publication Date (Web): 12 Apr 2018 Downloaded from http://pubs.acs.org on April 16, 2018
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Langmuir
Rheology of ultra-swollen bicontinuous lipidic cubic phases Chiara Speziale1, Reza Ghanbari1, Raffaele Mezzenga1,2* 1
ETH Zürich, Department of Health Science & Technology, Schmelzbergstrasse 9, 8092
Zürich, SWITZERLAND 2
ETH Zürich, Department of Materials, Wolfgang-Pauli-Strasse 10, CH- 8093 Zurich,
SWITZERLAND
Abstract Rheological studies of liquid crystalline systems based on monopalmitolein and 5 or 8% of 1,2 distearoylphosphatidylglycerol are reported. Such cubic phases have been shown to possess unusually large water channels, due to their ability of accommodating up to 80 wt% of water, a feature that renders these systems suitable for crystallizing membrane proteins with large extra-cellular domains. Their mechanical properties are supposed to be substantially different from those of traditional cubic phases. Rheological measurements were carried out on cubic phases of both Pn3m and Ia3d symmetries. It was verified that these ultra-swollen cubic phases are less rigid than the normal cubic phases, with the Pn3m being softer that the Ia3d ones. Furthermore, for the Pn3m case, the longest relaxation time is shown to decrease logarithmically with increasing surface area per unit volume, proving the critical role of the density of interfaces in establishing the macroscopic viscoelastic properties of the bicontinuous cubic phases.
Introduction Bicontinuous lipid cubic phases (LCPs) arise from the spontaneous self-assembly of specific lipids in water under defined conditions of temperature and composition. These are highly ordered and thermodynamically stable systems, in which interfaces are centered on triply
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periodic minimal surface (TPMS)1,2. There are three basic minimal surfaces, the gyroid (G), the diamond (D) and the primitive (P) surface, corresponding to Ia3d, Pn3m and Im3m crystallographic space groups, respectively. Their mean curvature at the TPMS position is always zero and the lipid bilayer, highly curved in three dimensions3, is surrounded by two identical, but nonintersecting aqueous channels4, with a three-fold, four-fold and six-fold symmetry, for the gyroid, diamond and primitive symmetry, respectively. LCPs are liquid crystalline systems in which the molecules are mobile (“liquid-like”), but diffusion is allowed only within a defined geometrical order, characteristic of a crystalline solid. Bicontinuous cubic phases have been observed for different types of amphiphiles5,6 and cubic membranes have also been observed in many eukaryotes cell types7,8. One of the features that renders LCPs so fascinating is that they can coexist at the equilibrium with excess water9,10; moreover, because of their amphiphilic nature and large surface area per volume, they are able to encapsulate an extensive amount of both hydrophilic and hydrophobic molecules11–15. Furthermore, these transparent and optically isotropic gels show striking viscoelastic properties that are comparable to cross-linked polymer networks. All these unique aspects make them an attractive tool for a number of applications, ranging from pharmaceutics and diagnostics16–19, to food science20 and cosmetics. LCPs also provide an excellent matrix for the entrapment of proteins21–24 and enzymes25,26. They have been also used as biosensors27, but mostly for membrane protein crystallization28,29. Cubic mesophases represent in fact an excellent environment for the protein to embed, reconstitute and crystallize within the lipid bilayer of the cubic phase. Proteins have been shown to maintain their structural integrity, to reconstitute correctly and therefore retain their biological activity30. Furthermore, the mesophase structure allows for the partial or complete mobility of the protein molecules, which at the same time helps the diffusion of protein molecules towards the growing crystal. Membrane protein crystallization from lipid cubic mesophases has recently revolutionized membrane structural biology, yielding several highresolution X-ray structures over the past two decades. One of the major restrictions to the use of these materials is represented by relatively small unit cells resulting in narrow water channels (3-5nm)31. Finding strategies to expand unit cells without compromising periodicity and ordering is a major challenge that a number of scientists are seeking to overcome.32–35 Most of the strategies undertaken so far are based on the addition in the LCP formulation of an additive which could swell the host lipid mesophase while preserving the bicontinuous cubic phase symmetry13,23,32,36. Usually, the additive consists of a small amphiphile which
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through electrostatic interactions with the lipid-forming cubic phase can increase the lattice parameter of the mesophase. We have recently demonstrated37 that thermodynamically stable ultra-swollen cubic phases were formed by adding in small amounts of an anionic phospholipid (1,2 distearoylphosphatidylglycerol, DSPG) to cubic phases based on monopalmitolein (MP), a cis-monoacylglycerol with a C18 carbon chain. Furthermore, membrane proteins with large extra-cellular domains were successfully crystallized within these new systems. It is important to note that such cubic phases can incorporate up to 80 % of water. Therefore, they are expected to exhibit unusual mechanical properties for bicontinuous cubic phases. The bulk rheology of lyotropic liquid crystals has been documented in a number of contributions38–44, and it shows an interesting complex behavior and viscoelastic properties. Cubic phases have been shown to be the most rigid liquid crystalline phases, followed by the reversed hexagonal phase, which is a moderately viscoelastic fluid, and the lamellar phase which can been described as a plastic fluid. In this work, we studied the rheological behavior of MP/DSPG cubic phases of Pn3m and Ia3d symmetry, obtained varying the amount of DSPG (5 and 8%) and water in the system. In particular, we focused on the study of the storage, G′, and loss moduli, G″ as a function of the angular frequency and on the longest relaxation time, τmax, to analyze the effect of the structure of such ultra-swollen cubic phases on their mechanical properties. Finally, we also introduced small amounts of cholesterol as a third lipid component to study its effect on the rheological behavior of the swollen cubic phases. In fact, previous studies45 have demonstrated that cholesterol may play a major role in reconstitution and crystallization of some membrane proteins, hence the relevance to the present study.
Materials and methods Monopalmitolein >99% (M-219) was purchased from Nu-Chek Prep (Minnesota, USA). 1,2distearoyl-sn-glycero-3-phospho-rac-glycerol, sodium salt, >99% (DSPG, 560400), was kindly provided by LIPOID PG (Steinhausen, Switzerland). Cholesterol, >99% was purchased from Sigma Aldrich-Chemie. Sample preparation. Lipid mixtures were prepared co-dissolving the proper amount of each component (MP, DSPG and cholesterol) in chloroform/methanol in a 8:2 ratio. Solvent was completely removed by putting the solution first at the rotary evaporator and under high vacuum overnight afterwards. Mesophase samples were then prepared by mixing inside ACS Paragon Plus Environment
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sealed Pyrex tubes weighed quantities of pure MP (for the control samples) or MP/DSPG(/chol) and Milli-Q water in order to obtain the desired percentage of hydration in each sample. The samples were then first vortexed at room temperature until a homogenous mixture was obtained and then centrifuged for 15 min at ∼2000g. The prepared mesophase was then allowed to equilibrate at room temperature for 24 hours. Small-Angle X-ray Scattering. SAXS measurements were used to identify the symmetry of the mesophases at the different conditions. Experiments were performed on a Bruker AXS Micro, with a microfocused X-ray source, operating at voltage and filament current of 50kV and 1000µA, respectively. The Cu Kα radiation (λCu Kα = 1.5418 Å) was collimated by a 2D Kratky collimator, and the data were collected by a 2D Pilatus 100K detector. The scattering vector q = (4π/λ)sinθ, with 2θ being the scattering angle, was calibrated using silver behenate. Data were collected and azimuthally averaged using the Saxsgui software to yield onedimensional intensity versus scattering vector q, with a q range from 0.005 to 0.4 Å−1. In addition, SAXS measurements were also performed on a using a microfocused Rigaku, X-ray source, of wavelength λ = 1.54 Å, operating at 45 kV and 88 mA. Diffracted X-rays signal was collected on a gas-filled two-dimensional (2D) detector. The scattering vector q = (4π/λ)sin θ, with 2θ being the scattering angle, was calibrated using silver behenate. Data were collected and azimuthally averaged using the Saxsgui software to yield 1D intensity versus scattering vector q with a q range from 0.003 to 0.3 Å−1. For all measurements, ∼200 µL of sample were loaded into 2 mm quartz capillaries and sealed with epoxy glue. Measurements were performed at 20°C, samples were equilibrated for 30min before measurements, whereas scattered intensity was collected over 60 min. Rheological Measurements. A stress controlled AR 2000 rheometer (TA Instruments) was used in cone-plate geometry, 2◦ angle, 20 mm diameter. Temperature control was set at 20°C. First a strain sweep was performed at 1 Hz between 0.008% and 200% strain to determine the linear range. Then oscillatory frequency sweeps were performed at 0.1% strain between 0.013 and 628 rad/s. To minimize solvent evaporation, a solvent trap was used whosesealing cavity filled with low-viscosity silicon oil.
Results and Discussion As it has been recently shown from our group37, incorporating a relatively small amount of DSPG, an anionic phospholipid, into MP-based cubic phase, leads to the formation of LCPs . Such LCPs are able to retain large amounts of water, up to 80%, in the bulk phase. They are ACS Paragon Plus Environment
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therefore characterized by remarkably large lattice parameters, up to five-fold compared to typical lipid mesophases32,33. It is interesting to note that such large lattice parameters have been also observed in biological cubic membranes. In this work we used two different lipid compositions to form bicontinuous cubic phases of either Pn3m or Ia3d symmetry: MP:DSPG were mixed in ratios of 95:5 or 92:8 and water contents ranging from 60 to 80 % were used when mixing with the lipid blend. As control samples, LCPs containing only MP and water were prepared: MP:water in a ratio 50:50 for the Pn3m cubic phase and MP:42 for the Ia3d one. According to the phase diagram of MP 46, these two samples corresponds to the maximum hydration conditions for the two LCPs. All the sample were characterized by SAXS measurements in order to assess the phase symmetry and determine the structural parameters (Figures S1-S3).
Figure 1. SAXS intensity profiles for a) Pn3m and b) Ia3d symmetry cubic phases at the maximum hydration conditions for each lipid composition considered. For the Pn3m case, the pure MP cubic phases contains 50% of water, while the DSPG-doped one 80%. For the Ia3d cubic phases, the pure MP based one contains 42% of water, while the DSPG-doped one 80%.
Figure 1 shows the difference observed in the SAXS intensity profiles between the two MPbased control LCPs of both Ia3d and Pn3m symmetry and the corresponding ones for the most swollen DSPG-doped cubic phase obtained with the same symmetry (containing 80% of water in both cases). As it can be seen, the phospholipid-containing cubic phases gave SAXS profiles shifted towards very low q values, which implies a tremendous increase in the lattice parameters (see Table 1).
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Table 1. Phase Symmetry, Unit Cell Parameters and Relaxation times obtained for each sample analyzed. Lipid Composition
% Water
Symmetry
Unit Cell (Å)
τmax (s)
τ7 (s)
MP
50
Pn3m
102
--
440.05
MP
42
Ia3d
145
--
760.60
MP:DSPG 95:5
55
Pn3m
125
71.43
--
MP:DSPG 95:5
60
Pn3m
145
62.50
--
MP:DSPG 95:5
80
Pn3m
320
20.08
28.30
MP:DSPG:chol 94.75:5:0.25
60
Pn3m
142
31.25
--
MP:DSPG:chol 94.5:5:0.5
60
Pn3m
143
25.02
--
MP:DSPG 92:8
60
Ia3d
256
598.80*
--
MP:DSPG 92:8
66
Pn3m
170
56.24
MP:DSPG 92:8
70
Pn3m
199
50.03
77.36
MP:DSPG 92:8
80
Ia3d
513
126.42
162.72
*This value was obtained by a linear extrapolation of the G’, G’’ vs shear frequency curves, since the crossover was not reached within the experimental frequency range.
Rheological measurements have been utilized to understand the properties and structure of such self-assembled systems. The presence of significant amounts of water renders them an interesting system to study from this point of view, since conventional LCPs never reach similar levels of water in their composition. By increasing the water in the system, less viscous cubic phases are obtained. We were interested in investigating the mechanical properties of MP/DSPG based cubic phases, expecting unusual behavior for bicontinuous cubic phases, due to their remarkable structural features. Measurement of the storage G’ and loss G’’ moduli as a function of the shear frequency, ω, can reveal important information about the characteristic behavior of viscoelastic materials. Frequency sweep measurements were performed at a constant strain in the linear viscoelastic regime, as determined by the oscillation strain sweep measurement performed for each sample ACS Paragon Plus Environment
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(Figure S4-S6). Within the linear viscoelastic region in fact, the material response is independent of the magnitude of the deformation and the material structure is maintained intact; this is a necessary condition to accurately determine the mechanical properties of the material.
Figure 2. Storage, G′ (filled symbols), and loss, G′′ (empty symbols), moduli as a function of shear frequency of Pn3m cubic phases composed of a) MP, 50% water; b) MP:DSPG 95:5, 55% water; c) MP:DSPG 95:5, 60% water; d) MP:DSPG 92:8, 66% water; e) MP:DSPG 92:8, 70% water; f) MP:DSPG 95:5, 80% water.
Figure 2 shows the G’ and G’’ curves vs ω for different Pn3m cubic phases obtained by varying the ratio MP/DSPG and the water %. If we consider the general viscoelastic behavior for a structured fluid47,48, in all cases the region of the so-called rubbery plateau is visible, with G’>G’’. Furthermore, at progressively low frequencies the curves undergo the transition to flow towards the viscous regime, which corresponds to the region after the crossover of the two curves, when G’’ becomes lower than G’. The spectra of G′ and G″ versus shear frequency, also allow extracting the longest relaxation time, τmax, defined as the inverse of the frequency at which crossover of G′ and G″ takes place. τmax is the characteristic time at which the structured fluid relaxes back to the equilibrium configuration after being perturbed. The physical meaning of this relaxation time in LCPs is generally attributed to the diffusion time of the molecules of lipid at the water-lipid interface41. As it can be observed, by increasing the amount of water in the system and consequently the lattice parameter of the resulting LCP, ACS Paragon Plus Environment
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both the storage and loss moduli decrease in the range of angular frequency considered and the crossover of the two curves, which is not reachable for the pure MP/water system, appears at progressively higher frequency values, implying a decrease in the longest relaxation time.
Figure 3. Storage, G′ (filled symbols), and loss, G′′ (empty symbols), moduli as a function of shear frequency of Ia3d cubic phases composed of a) MP, 42% water; b) MP:DSPG 92:8, 60% water; c) MP:DSPG 92:8, 80% water.
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Figure 3 shows the storage and loss moduli as a function of the shear frequency for the Ia3d cubic phases. Overall, a similar behavior is observed, nevertheless some differences can be underlined as well. In particular, LCPSs of Ia3d symmetry show higher values of the storage modulus at fixed amounts of water (cfr. Fig. 2c with Fig. 3b and Fig. 2f with Fig. 3c), sign of higher strength and mechanical rigidity of the sample with respect to the Pn3m ones. Furthermore, in the case of Ia3d cubic phase, the crossover of G’ and G’’ happens at frequencies much lower than in the case of the Pn3m cubic phases having the same water content. The viscoelastic behavior of LCPs can be further described by a Maxwell fluid, where the storage and loss moduli read:
=
1 1 +
=
2 1 +
= 3 where and stand for zero-shear rate viscosity and instantaneous shear storage modulus, respectively and represents . However, as previously demonstrated in the literature,
LCPs exhibit complex rheological behavior40,49. Such complex rheological behavior can be better captured by a multiple Maxwell fluid where frequency dependence of storage and shear moduli are expressed as:
=
=
4 1 + 5 1 +
In these equations, n is representative of the number of spring and dashpot pairs. Each relaxation mode corresponds to th spring and dashpot pair which is characterized by , th
zero-shear rate viscosity and , th relaxation time. The more relaxation modes are, better the material functions of the cubic phases are described.
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Figure 4. Storage, G′ (empty symbols), and loss, G′′ (filled symbols) moduli as a function of shear frequency for a) MP, 50% water (circle), MP:DSPG 92:8, 70% water (square), MP:DSPG 95:5, 80% water (diamond); b) MP, 42% water (circle), MP:DSPG 92:8, 80% water (square); ( , ) pairs for c) MP, 50% water (circle), MP:DSPG 92:8, 70% water (square), MP:DSPG 95:5, 80% water (diamond); d) MP, 42% water (circle), MP:DSPG 92:8, 80% water (square)
Figure 4a and 4c show fits of experimental storage and loss moduli of controlled and DSPGdoped LCPs with Pn3m and Ia3d symmetries, respectively, while Figure 4b and 4d present the spectra of zero-shear rate viscosity with corresponding relaxation time for each fit. As previously mentioned by Mezzenga et al, irrespective of the number of modes, the highest relaxation mode in a multiple Maxwell fluid is attributed to the relaxation of lipid-water interface40. The rest of the modes acquired from seven-mode Maxwell fluid can be attributed to relaxation of the lipid chains, confined water and other polydispersities in the system.
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Conclusively, the dominant relaxation time, i.e., the highest relaxation, extracted from
fitting data agrees well with in terms of order of magnitude (see Table 1). In the case of
control MP-based cubic phases, the seven-mode Maxwell fluid estimates the highest relaxation time of 440.05 s and 760.60 s for Pn3m and Ia3d cases, respectively, whereas due to experimental limitations, the frequency at which and overlaps, was not captured in
these cases. Furthermore, similar to , value is shifted to lower values for swollen LCPs compared to undisturbed ones. More importantly, the general trend observed in all the discrete spectra in Figure 4b and 4d is that upon increasing hydration, the individual weight of each relaxation time decrease, reflecting a softening of the system.
As already mentioned, the Ia3d cubic phase showed an enhanced rigidity compared to the Pn3m case. This could be due to the different 3D organization of the lipid bilayer in the two symmetries; in particular, because of its topological characteristics (three-fold symmetry of the water channels and the compactness of the minimal surface), the Ia3d cubic phase is the least porous50. To this point it is worth to analyze how τmax varies when comparing –at fixed amounts of water- two samples with different symmetry (Pn3m and Ia3d) but with the same unit cell surface area per unit volume (A/a3). To obtain the unit cell surface area (A) at the interface between lipid and water one has first to calculate the length of the lipid chain, Llip following Turner et al.51, = 2
!"#$
% + 2&'
!"#$ (
%
(6)
where is the lipid volume fraction, a is the lattice parameter as measured by SAXS and A0 and χ have the following values: A0=3.091 and '=-8 for Ia3d and A0=1.019 and ' =-2 for Pn3m. Having computed the value of Llip, one can calculate the surface area per repeat unit following Anderson at al.2, ε = * + 2&'+ (7) where ε is the distance to the pivotal surfaces from the chain termini that is considered to be equal to Llip at the minimal surface. The resulting pivotal surface per repeating unit, needs then to be divided for the cube of the lattice parameter to obtain the unit cell surface area per unit volume (A/a3). This
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normalization -which apart from a multiplicative density prefactor is equivalent to the specific area in porous materials- is necessary in order to compare two intensive quantities. For the Pn3m samples with 60% and 80% of water we have values of A/a3 equal to 0.01233 and 0.005911 and τmax 62.50 and 20.08 s, respectively. In the Ia3d case, while A/a3 is practically the same, (0.01121 for the sample with 60% of water and 0.005922 for the 80% one) τmax is considerably longer (598.80 and 126.42 s, respectively). Moreover, Saksena et al.
52
conducted
simulations on ternary amphiphilic systems of Ia3d, Pn3m and Im3m and comparing the rheological responses of the three cubic phases, found the Ia3d system being the most viscous. This means, that at identical specific area, the structure resulting from the organization of different space groups within distinct symmetries, still plays a major role on establishing the final rheological properties. Next, we studied how, for a fixed symmetry, the rheological fingerprint varies with the topological characteristics. Thus, for the Pn3m case, which is available in a sufficiently large window of compositions, the dependence of τmax on the structural parameters of the LCP was studied.
Figure 5. Longest relaxation time as a function of the surface area per unit volume of the five cubic phases of Pn3m symmetry composed of MP and DSPG. The inset shows how τmax varies with the ln of the area per unit volume. Figure 5 shows the plot of τmax as a function of A/a3. As it can be observed, there is a logarithmic dependence of the relaxation time from the normalized surface area. This can be better visualized from the inset of Figure 5, where a linear fit describes the plot τmax vs ln(A/a3). Frequency sweeps were also conducted on samples containing a small amount of cholesterol in the lipid mixture. The Pn3m LCP sample containing 60% of water and MP:DSPG in a ratio
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95:5 was used as control sample. We then prepared LCPs containing the same amount of water, but adding small amounts of cholesterol (while decreasing the MP fraction) to obtain samples with the same phase symmetry and lattice parameters. These conditions were fulfilled by the lipid mixtures MP:DSPG:chol in ratios 94.75:5:0.25 and 94.5:5:0.5.
Figure 6. Effect of cholesterol on the rheological properties of MP/DSPG based cubic phases. G′ (filled symbols) and G′′ (empty symbols) vs shear frequency for a) MP:DSPG 95:5, 60% water and in presence of b) 0.25% and c) 0.5% of cholesterol. d) Decrease of the longest relaxation time with increase of the amount of cholesterol. Errors bars are not shown as they represent at most 0.5% of the averages.
Figure 6 shows the G′ and G″ versus shear frequency curves for these two samples compared to the LCP without cholesterol. As also summarized in Figure 6d, although the storage and loss moduli remain unchanged in presence of cholesterol, the crossover appears at larger angular frequencies, meaning the τmax is shorter and that the cubic phase undergo the transition to the viscous regime earlier; in other terms, cholesterol is found here to have a counterintuitive “softening” effect. Cholesterol is known to affect in different ways lipid ACS Paragon Plus Environment
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bilayers. Extensive studies have been made on phospholipid-based bilayer, since they are the main components of cell membranes, where cholesterol is present up to the 30% wt of the lipid fraction53. While it helps the lipid chains in the bilayer to pack close and ordered, decreasing the permeability of the system54, it has also been reported that the higher complexity (compositional and distributional) derived from the presence of a different lipid with a rigid hydrophobic moiety, should result in a higher correlation of the motion in the membrane, and this account for a higher fluidity55. Cholesterol influence on lipid bilayers of monoacylglycerols-based LCPs has also been investigated as it represents a lipid additive used for in meso crystallization assays. In particular, cholesterol was found to swell up to 15 Å the water channels of Pn3m bicontinuous cubic phases composed of monoolein, a monoacylglycerol similar to MP, but with a C18 lipid chain instead of C1636. Moreover, beyond 23 mol %, the presence of cholesterol causes a change in the phase symmetry from Pn3m to Im3m. This effect might be explained by the alteration of the curvature at the lipid-water interface caused by cholesterol through increased hydration of the head groups32,36,56. Taken together, these observations are in agreement with our experimental findings that show , although a very small amount of this lipid in the bilayer of the LCP does not alter further the structure, it renders the bilayer more fluid. As a result, the gel starts acting as a viscous fluid at higher frequencies, and its longest relaxation time becomes shorter. Finally, it should be also noted that all the investigations found in the literature are focused on fractions of cholesterol in the bilayer two orders of magnitude larger compared to this work.
Conclusions In this work, we have studied the rheological behavior of ultra-swollen cubic phases. The cubic
phases
composed
of
a
lipid
blend
of
monopalmitolein
and
1,2
distearoylphosphatidylglycerol can incorporate large amounts of water, resulting in remarkably large unit cell dimensions. As expected, these LCP showed interesting mechanical behaviors, due to their unique structural features. We studied cubic phases of Pn3m and Ia3d symmetries, with the Ia3d samples being more rigid than the Pn3m ones. For all the 1,2 distearoylphosphatidylglycerol-doped system we were able to calculate the longest relaxation time and study how this relate to the topological features of the mesophases. For both cubic phases’ symmetries, τmax was decreasing with increasing the lattice parameter and the water ACS Paragon Plus Environment
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content, while comparing Pn3m and Ia3d cubic phases with same water content, τmax was found to be longer in the case of Ia3d cubic phase. Similar results were extracted from fits using a multiple Maxwell fluid model where we observed a good agreement with experiments. We attribute this finding to the structural differences between the two symmetries, in particular to the more compact arrangement of the 3D minimal surfaces of the lipid bilayer of the Ia3d cubic phase, with respect to the Pn3m symmetry, which leads to smaller water channels. Indeed, for the Pn3m ultra-swollen lipid cubic phases we found a logarithmic dependence of the τmax on the specific area of the bicontinuous cubic phases, obtained as the total area per repeat cell divided the corresponding repeat cell volume. This immediately highlights the pivotal role of the density of interfaces in establishing the macroscopic viscoelastic properties of the bicontinuous cubic phases. Finally, by adding small amounts
of
cholesterol
to
the
lipid
composition
of
the
monopalmitolein/1,2
distearoylphosphatidylglycerol cubic phases, we observed a further effect on the relaxation time, which decreased with increasing the cholesterol fraction, pointing at a fluidifying role of this rigid molecules in the periodic lipid mesophases.
ASSOCIATED CONTENT The Supporting Information file includes Supplementary Small angle X ray scattering diffractograms and shear rheology linear regime identification. AUTHOR INFORMATION Corresponding Author * Prof. Dr. Raffaele Mezzenga, ETH Zurich, Department of Health Science & Technology, Schmelzbergstrasse 9, LFO, E23, 8092 Zürich, SWITZERLAND. Tel: +41 44 632 91 40. Email:
[email protected] ACKNOWLEDGEMENTS
We gratefully acknowledge Dr. Salvatore Assenza for valuable discussions. This work was supported by the Swiss National Science Foundation (NSF) Sinergia Grant CRSII2_154451.
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