Impact of Topology on the Characteristics of Water inside Cubic

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Impact of Topology on the Characteristics of Water inside Cubic Lyotropic Liquid Crystalline Systems Konoya Das, Bibhisan Roy, Sagar Satpathi, and Partha Hazra J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.9b01559 • Publication Date (Web): 22 Apr 2019 Downloaded from http://pubs.acs.org on April 23, 2019

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The Journal of Physical Chemistry

Impact of Topology on the Characteristics of Water inside Cubic Lyotropic Liquid Crystalline Systems Konoya Das,ǁ Bibhisan Roy,ǁ Sagar Satpathiǁ and Partha Hazra*,ǁ,ǂ

ǁDepartment

of Chemistry, Indian Institute of Science Education and Research (IISER), Pune

Dr. Homi Bhabha Road, Pashan, Pune, India 411008. Fax: +91 20 2590 8186 ǂCentre

for Energy Science, Department of Chemistry, Indian Institute of Science Education

and Research (IISER), Pune, India 411008 E-mail: [email protected], Tel: +91 20 2590 8077

1 ACS Paragon Plus Environment

The Journal of Physical Chemistry 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

ABSTRACT: Water molecules present inside the lipid based cubic liquid crystalline phases are found to play a major role in wide range of applications, such as, protein crystallization, virus detection, delivery of drug and biomolecules, etc. In this regard it is crucial to elucidate static and dynamic properties of the water molecules in the nanochannels and explore the effect of geometrical topology on the nature of the water inside the different cubic phases. In the present work, we have incorporated two probes, coumarin-343 (C-343) and coumarin-480 (C480) in two cubic phases with different symmetries namely gyroid (Ia3d) and double diamond (Pn3m) with the same water content (22%), to probe the micropolarity, microviscosity and the hydration dynamics at different hydrophobic depths in the mesophases. Steady state results estimate the polarity at the lipid-water interface to be similar to ethanol, and the polarity near the more hydrophilic parts of the nanochannel resembles ethylene glycol. We have also observed a gradient in the microviscosity inside the LLC nanochannels from time-resolved fluorescence anisotropy studies. The hydration dynamics, which play a key role in the numerous applications of the mesophases, has been probed by the time-dependent stokes shift method of the two probes revealing the existence of three kinds of dynamics. The difference in the hydration dynamics inside the two mesophases, where the water molecules confined in Ia3d phase exhibit a slower dynamics compared to that in Pn3m, is the prime importance of this work. The underlying reason for this disparity is majorly associated to the differences in the topology of the two structures including the hydrophobic packing stress, negative interfacial curvature and the curvature elastic energy of the lipid-water interface. We believe that this kind of correlation between the structural topology of the different cubic LLC mesophases and nature of water nanochannel will help to boost the applications of the cubic phases in the future.


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INTRODUCTION Lyotropic liquid crystalline (LLC) ‘soft’ materials have emerged as a promising class of substance due to their utilization in a wide range of applications such as nanoreactors for protein crystallization,1, 2 drug and nutrients delivery,3-5 food technology,6, 7 etc. Generally, unsaturated monoglyceride lipids, for instance, glyceryl monooleate (GMO) and monolinoleate (GML), are chosen for the synthesis of LLC mesophases owing to their biocompatibility and exceptional phase behavior.8,

9

LLC materials of different characteristic topologies and

symmetries, such as the 1D inverse hexagonal phase (HII), the 2D planar lamellar phase (Lα) and the 3D bicontinuous cubic phases (V2) have been synthesized by controlling the content of amphiphile or water in the system and the temperature.10 The fluid lamellar phase consists of amphilphilic bilayers separated by water domains and closely resembles the cellular membranes.6 The HII phase is a topologically inverted “water in oil” system comprising infinitely long and straight water filled rods packed densely in a non-polar matrix of lipohilic chains.6, 11 The inverse bicontinuous cubic phases namely, gyroid (Ia3d) and diamond (Pn3m)

Scheme 1. Schematic representation of cubic LLC phases (Ia3d and Pn3m). 3 ACS Paragon Plus Environment


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are formed by the arrangement of a continuous lipid bilayer on mathematical minimal surfaces (Scheme 1).12, 13 In Ia3d mesophase two water networks join in a 33 junction at an angle of 120° and in Pn3m they join in a 44 manner at a tetrahedral angle of 109.5°.13 Larsson was the first to suggest that the infinitely periodic continuous structure of the lipid membrane in the cubic phases closely resembles that of lipidic membranes in nature.14 Consequently, the biological aspects of the cubic phases have garnered significant attention among researchers owing to their roles in membrane fusion, control of membrane protein functions and various intracellular structures of membranes.15, 16 The unique structural features of the cubic phases allow the transport of both hydrophilic and lipophilic targets, and the phases have been utilized extensively for the in vitro and in vivo delivery of drugs and oligopeptides.17, 18 In the last decade the cubic phases have been used to facilitate the crystallization of membrane proteins inside its nanochannels which do not tend to crystallize in bulk1, 19, 20 and for the detection of viruses and bacteria.21 The nature of nanochannels, dynamics of the water molecules and interfacial hydration is proposed to play an important role behind the crystallization process.22 CH3

O OH N

O C-343

N

O

O

O

C-480 O

O

OH

HO GML

Scheme 2. Chemical structures of coumarin-343 (C-343), coumarin-480 (C-480) and 1Linoleoyl-rac-Glycerol (GML).


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Water confined in a small volume of supramolecular assemblies is fundamentally different from bulk water.23, 24 Unravelling the dynamics of water in the biologically pertinent assemblies is crucial for understanding how the confined water influences the structure, reactivity, molecular recognition and dynamics in a biological interface. An important discovery is the two to four-fold slower dynamics of water in restricted assemblies compared to bulk water.

23, 24

Considering the important applications of the LLC phases based on the

confined water, our group has recently investigated the microenvironment inside the narrow channels of HII phase using coumarin-343 probe.25 The study of solvation dynamics exhibited the existence of two types of water molecules inside the nanochannels. Dielectric relaxation spectroscopic studies of the reverse hexagonal phase corroborate this observation.26, 27 It was shown that the dynamics of a large percentage of water molecules were slowed down due to interactions with the lipid interface and the remaining water located away from the interface adopts bulk water like behavior. Importantly, Zhong et al. have mapped femtosecond scale hydration dynamics at different depths of the GMO based Pn3m cubic mesophase using a series of tryptohan alkyl esters.28 They have reported three different dynamics denoting discrete water structures, namely, 100-150 ps for the interfacial water at the lipid surface, 10-15 ps for the adjacent layer of hydrogen bonded water molecules and < 1 ps for bulk like water at the centre of the channel. Although tryptophan is an attractive solvation probe, it may only be used to detect ultrafast solvation components because of the existence of two rotamers having different lifetimes giving rise to an apparent dynamic spectral shift in a few hundred ps timescale.29 Consequently, the major slow hydration components of the confined water remain undetected in the cubic LLC phases. In addition, the correlation of the structural disparities between the two cubic phases, Ia3d and Pn3m, to the behavior of water molecules confined in the nanochannels has not been explored. Inspired by the relevance of the LLC water networks and the absence of any detailed studies for the cubic mesophases, we have probed the dynamics of



the confined water molecules by the time-dependent Stokes shift method. In order to probe the dynamics of water molecules residing at the different depths of the nanochannels, we have selected two polarity sensitive coumarin probes (Scheme 2), namely, coumarin 343 (C-343) and coumarin 480 (C-480). We have qualitatively assessed the micropolarity of the various water layers in the two phases by utilizing the sensitive solvatochromic behavior of the coumarin molecules. We have also for the first time, quantitatively measured the microviscosity at different regions of the nanochannels, underlining the vast differences in the microviscosity for the two similar cubic phases. Although previously the two cubic phases are considered to be similar in nature, our observations highlight that the behavior of water in the nanochannels are essentially different in the light of their hydration dynamics and viscosity. We believe these new insights will help to boost the wide scale applications of these phases in different fields.

EXPERIMENTAL SECTION Materials.1-Linoleoyl-rac-glycerol (GML, purity 97%), coumarin 343 (97%) and coumarin 480 (99%) were purchased from Sigma-Aldrich and used as received. Milli-Q water was used for loading the dyes inside the Pn3m and Ia3d mesophases. Preparation of the Dye loaded LLC Phases. GML based Ia3d and Pn3m phases containing 22% water were prepared following the phase diagram reported by Mezzenga and co-workers.7 For the synthesis of each phase, GML was first heated at the required temperature of the particular phase for a few minutes. The temperatures required for Pn3m and Ia3d phases at 22% water are 55 °C and 45 °C, respectively. The coumarin dyes were dissolved in milli-Q water separately and the concentration of the dyes were adjusted to 8 M. The water containing the dyes was heated to remove any dissolved oxygen. The preheated water containing the dyes was then added to the liquid GML at constant stirring. The mixture was then stirred for around 6 ACS Paragon Plus Environment

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15 minutes and the required temperature for the respective phase was maintained throughout the process. The complete preparations were done in inert conditions to avoid the oxidation of GML. Subsequently, each sample was cooled to 25 °C and allowed to equilibrate for 48 h before the spectroscopic experiments. All the prepared LLC phases were optically transparent gel-like material and were characterized using polarized optical microscopy and small-angle X-ray scattering (Note S1). Instrumentation. Steady State and Time Resolved Fluorescence Measurements. All the steady state and time resolved fluorescence measurements were done using a solid state sample holder, with the gellike transparent cubic phases placed on a quartz slide, at a 450 with respect to incident light. During the measurements, the humidity of the room was maintained 10 ns) are probed by the C-480 molecules. This observation concurs with the steady state results, where the more hydrophobic C-480 probe is deemed to reside at the deeper layers of the nanochannels, and hence, the contribution of the ‘pseudo-bound water’ water layers are absent in the components of hydration of this probe. Notably, the self-diffusion of C-480 has a significant contribution towards the slower hydration dynamics in both Ia3d and Pn3m phases, which is reflected from the appreciable decrease in the FWHM of the TRES profiles at longer times. Additionally, the hydration dynamics slows down significantly in the Ia3d phase in comparison with the Pn3m phase. Although this has also been observed for C-343, the effect is more highlighted for the C-480 probe. An important point to note here is that the dynamics of the bulk like water is very similar in both Ia3d and Pn3m phases and the difference in the hydration dynamics between the two phases arises mainly at the longer time scales. While it is crucial to understand and assign the different categories of water molecules in the nanochannels, the principle motivation of this work is to interpret the differences in the microenvironment and hydration dynamics of the two cubic phases, Ia3d and Pn3m. We anticipate that the curvature of the lipid bilayers as well as geometry of the phases is the key to


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rationalize the observed differences of the water dynamics between the Ia3d and Pn3m nanochannels. According to the bimodal theory of the dynamic exchange between the ‘bound’ and ‘free’ water molecules,53,

54

inter-conversion between these molecules is the rate

determining step in solvation dynamics, which is governed by the diffusion of the water molecules. Here, it is assumed that the ‘free water molecules’ are free to rotate and the ‘bound water molecules’ can rotate only in restricted cooperation with the binding system. In the Ia3d phase, the two water channels have trifold connectivity (120°) whereas, the Pn3m phase has tetrafold connectivity (109.5°).56 From these geometrical characteristics of the two phases it can be reasoned that the tetrahedral connection angle of the water channels in the Pn3m phase would provide a faster pathway for the diffusion of ‘free/pseudo-bound’ water molecules compared to the trifold connected channels of the Ia3d phase leading to a slower water dynamics in the latter phase. Apart from the diffusion-controlled exchange of bound and free water molecules, the slow hydration component has also been determined to arise from the self-diffusion of the probes, particularly for C-480. It is expected that the diffusion of the probe is also more facile in the water channels of Pn3m phase. This behavior has also been mirrored in the literature report of diffusion controlled enzyme kinetics in the LLC matrices.57 It is seen that at the same channel size, the tetrafold geometry of Pn3m provides the fastest diffusion of substrates to the entrapped enzymes active sites, and subsequent products also diffuse away fastest, due to the tetra-fold connectivity.57 On the other hand, some recent reports suggest that the diffusion of point like molecules will be faster in the Ia3d phase compared to Pn3m owing to the lesser presence of bottlenecks in the former structure.58, 59 However, from the results, it is evident that the dynamics of pseudo-bound water molecules are very similar in both Ia3d (0.16 ns) and Pn3m (0.14 ns) indicating that the diffusion behavior for these water molecules in both the phases is similar. Consequently, for the prepared Pn3m and Ia3d phases at the same water content (22%), the effect of water nanochannel connectivity leading to a difference in 17 ACS Paragon Plus Environment


the hydration dynamics between the two phases is minimum. We have also calculated the radius of the water nanochannels of both these phases (Note S3 in SI), which lies in the range 2 nm - 2.3 nm. The larger radius of the water nanochannels compared to the size of the water molecules also concurs with the observed minimum differences in the dynamics of the pseudobound water molecules in the two phases. Thereby, it can be concluded the overall slower hydration dynamics in the Ia3d phases compared to Pn3m is arising from some other structural factors rather than the water channel connectivity or radius of the two phases. The differences in the hydration dynamics of the two cubic phases majorly arises in the third component (small amplitude motion of lipid head groups) and to a lesser extent in the second component (water molecules trapped in the lipid bilayer region). This implies that the differences in Pn3m and Ia3d mainly arises from the geometrical aspects of the lipid-water interface region. In this regard, a salient feature is that the two cubic phases have subtle differences in their structural aspects with respect to the curvature elastic energy and the hydrophobic packing stress. The inverse bicontinuous cubic phases (Ia3d and Pn3m) having negative interfacial curvature (wherein the hydrophilic/hydrophobic interface curves towards the hydrophilic domain) are composed of a single continuous lipid bilayer draped on a minimal surface.60 The lipid bilayers have been simplistically considered as an infinitely thin elastic surface where the curvature elastic energy is the energy cost associated with deformations of the surface by changing the mean or Gaussian curvature.60 The hydrophobic packing stress is defined as the energy penalty required for deforming the lipid chain away from its preferred average shape.61 The packing stress, the curvature elastic energy and negative curvature follow the order, Ia3d > Pn3m.15,62 From the nanochannel radius calculations as well, it is evident that the Pn3m will have a higher negative value of Gaussian curvature as the two quantities are inversely related.58, 59

Consequently, compared to Ia3d, in Pn3m the spontaneous curvature of the lipid bilayer

towards water region decreases. This connotes that the hydrophilic groups of the lipid facing


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towards the channels are more closely packed in the Ia3d phase than that in the Pn3m phase. This leads to the restriction in the rotational mobility of the hydrophilic headgroups of the lipid molecules in the Ia3d phase compared to the Pn3m phase. This is evident in the increased value of the ultraslow component (>10 ns), which is due to the slow amplitude motion of lipid headgroups, in the Ia3d phase in comparison with the Pn3m phase for both C-343 and C-480. Notably, the effect was more apparent for the C-480 probe, which is localized more near the lipid regions than the C343 probe due to its higher hydrophobicity. As a result, the motion of the lipid polar groups probed by C-480 in the Pn3m phase, was ~9 ns which slowed down significantly to ~16 ns in Ia3d phase. Importantly, the curvature of the polar headgroups in turn also hinders the mobility of the water molecules bound to them (intermediate hydration component). It has been previously observed that in systems with higher curvature, a water molecule in the periphery cannot have simultaneous favourable interactions with all of the headgroups near it, which increases the energy barrier leading to slower dynamics of the bound/trapped water molecules. This kind of topological effect termed as “curvature-induced frustration”,63 is more pronounced in the Ia3d phase than the Pn3m phase, leading to the overall slower hydration dynamics in the Ia3d phase. To sum up the results of hydration dynamics, we have used two coumarin molecules of ranging hydrophobicity to probe the dynamics of water at different layers in the aqueous nanochannels of cubic LLC phases. C-343 exhibits fast, slow and ultraslow hydration components whereas C-480 only shows the latter two components. It is noteworthy to mention here that we have previously reported the solvation dynamics of C-343 in the HII LLC phase at the same water content and solvation components of similar time scale were obtained. We have anticipated the existence of two classes of water molecules in the nanochannels, where one hydration shell comprises of water molecules bound to the hydroxyl groups of the lipid. The cooperative motions of the lipid chains slow down the translational and rotational motions of



these bound water molecules and are accountable for the slow hydration dynamics. Another hydration shell comprises of the ‘pseudo-bound’ water and is responsible for the fast dynamics. These water molecules are hydrogen bonded to the bound water molecules which causes a restriction in their motion and hence they exhibit slower dynamics compared to bulk water. The ultraslow component is attributed to the small amplitude motion of the lipid headgroups. The dynamics of ‘pseudo-bound’ (~ 150 ps) water detected in our case is similar to the slowest component observed by Zhong et al. in the Pn3m phase which they had attributed to one of the layers of bound water.28 However, since tryptophan is unsuitable for detecting nanosecond dynamics, the contributions of the slow and ultraslow components have been completely missed in their study. Although the various aspects such as basicity of water confined in the nanochannels,22 structure-temperature relationship25,

27

surfactant dependent packing stress

etc16 of the different LLC phases have been documented, there is a stark lack in the comparison between the two cubic LLC phases, Ia3d and Pn3m in terms of the properties of the water molecules confined in the nanochannels. Considering the crucial applications of the two different cubic phases based on their nanochannels1, 17, 18, 20, 21 we have explored hydration dynamics in the Ia3d and Pn3m phases and elucidated in details the influence of curvature topology and geometry on the differences in the observed water dynamics between the two phases. Time-Resolved Fluorescence Anisotropy Study. The observed evidence of diverse water networks inside the LLC phases suggests that the local viscosity in the various regions of the nanochannels may also differ. In this regard, time-resolved fluorescence anisotropy measurement is considered to be an efficient technique to estimate the mobility of the probe molecule and hence predict its surrounding microviscosity (Figure 5). Both from the steady state as well as the hydration dynamics studies, it is apparent that C-480 is largely localized near the hydrophobic interfacial regions of the channels whereas, C-343 is more evenly


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distributed in the nanochannels of the LLCs. Thus, the rotational diffusion time constants of C-343 is expected to provide more thorough information regarding the microviscosity at various depths in the aqueous channels of the LLC phases. Subsequently, we have carried out the time resolved anisotropy measurements using C-343 in both the cubic phases, Ia3d and Pn3m, in order to understand the differences in microviscosities experienced by the coumarin probe as a result of the geometrical architecture of the cubic LLCs. The anisotropy transients of C-343 in both Pn3m and Ia3d phases exhibit triexponential decay with fast (~120 ps), intermediate (~ 1 ns) and slow nanosecond (~ 3 ns) components. Here it is necessary to mention that the rotational relaxation of C-343 in bulk water occurs at ~ 130 ps time scale. Thus, the observed fast ~125 ps time constant in the LLC phases suggests the rotational motion of the probe in the central part of the water channels, where the water exhibits a bulk like behavior. The ~ 1 ns component in both the phases is believed to be originated from the rotational relaxation of C-343 molecules partitioned in between the central ‘water pool’ and ‘interfacial region’. The observed slowest rotational relaxation component is credited to the coumarin probe partitioned near the interfacial regions of the nanochannels. The C-343 molecules in this region can take part in hydrogen bonding with the polar headgroups of the lipid and also with the ‘bound water’ molecules, which effectively hinders the mobility of the probe molecule. The slower rotational relaxation times of C-343 in the Ia3d phase compared to the Pn3m phase highlights the retarded mobility of the C-343 molecules due to its geometrical restrictions, such as, higher negative curvature of lipid bilayer, curvature elastic energy as well as hydrophobic packing stress which have been discussed in details in the previous section. The retarded relaxation dynamics in the Ia3d phase also points towards a higher microviscosity faced by the C-343 molecules in the nanochannels of Ia3d in comparison to those of Pn3m.



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Figure 5. Time resolved anisotropy measurements C-343 in both Pn3m and Ia3d phase. (λex = 402 nm and λem = 490 nm) The micro-viscosity in the different regions of the aqueous nanochannels of the two cubic phases has been estimated by the Debye-Stokes-Einstein equation,64 𝜂=

𝜏𝑟𝑘𝐵𝑇 𝑉

(4)

where, τr is the rotational relaxation time obtained experimentally, kB is the Boltzmann constant, T is the temperature in Kelvin scale and V is the volume of the probe molecule (considering a radius of 5.20 Å for C-343, see note S4 in SI). Using the fast-rotational relaxation time (~130 ps) of C-343, the calculated microviscosity for Pn3m and Ia3d phases turns out to be 0.87 and 0.91 respectively, which is similar to the micro-viscosity sensed by C-343 in bulk water, implying the existence of central ‘bulk water’ like region inside the nano-channels. The microviscosity calculated using ~ 1 ns rotational time constant is found to be 6.30 cP for Pn3m


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Table 2. Rotational Relaxation Dynamics Parameters of C-343 in Pn3m and Ia3d Phases Sample C-343 in water C-343 in Pn3m C-343 in Ia3d

r0

α1

τ1 (ns)

η1 (cP)

α2

τ2 (ns)

η2 (cP)

α3

τ3 (ns)

η3 (cP)

0.37

1.0

0.130

0.91

-

-

-

-

-

-

0.38 0.26

0.125

0.87

0.54

0.900

6.30

0.20

2.86

20.00

0.38 0.25

0.130

0.91

0.45

1.10

7.70

0.30

3.30

23.10

and 7.70 cP for Ia3d phases. This depicts the microviscosity sensed by C-343 molecules in regions between the central ‘water pool’ and lipid-water interfacial region of the cubic nanochannels. The microviscosity in the interfacial regions of the nanochannels have been calculated from the slowest component, with the interfacial regions of Ia3d exhibiting a higher microviscosity (23.10 cP) than Pn3m (20.0 cP). Two plausible factors can be put forth to explain the high microviscosity of the water confined in the LLC nanochannels, one being the different structure of the water in a confined geometry compared to bulk. The other contributing factor is the hydrogen bonding between the water molecules and the -hydroxy groups of the lipid which is mainly responsible for the drastic > 20-fold increase in the microviscosity of the interfacial water compared to bulk water. This kind of bonding renders certain ‘stiffness’ to the water molecules which has also been previously reported for other gel like materials.65 In a nutshell, time-resolved anisotropy studies provide insight about the existence of microviscosity gradient inside the LLC nanochannels, which is attributed to the variation of hydrogen bonded networks inside the nano-channels.

CONCLUSION The rationale behind this work was to elucidate the influence of the structural topology of the two cubic phases, Ia3d and Pn3m, which differentiate the nature of their respective 23 ACS Paragon Plus Environment


nanochannels with respect to micropolarity, microviscosity and the hydration dynamics. From steady state fluorescence and REES studies of two coumarin probes (C-343 and C-480), it is clear that hydrophobicity of the probes controls their distributions inside the nanochannels. Hydrophobic C-480 is lodged homogenously near the less polar lipid headgroups, whereas C343 is inhomogenously distributed nearer to the polar central core of the nanochannels. This gives an idea of micropolarity of the lipid-water interface similar to ethanol and ethylene glycol like polarity near the more polar central core. Utilizing the preferential location of the molecules, we have probed the hydration dynamics of the mesophases where, the ultraslow component has been ascribed to the small amplitude motion of the lipid headgroups. The slow component is assigned to the restricted motion of the water molecules bound to the hydrophilic headgroups and the fastest component to the ‘pseudo-bound’ water molecules which are hydrogen bonded to the bound water molecules. Previously reported hydration dynamics for the Pn3m phase did not include any slow components (> 150 ps) and more importantly any comparison with other cubic phases is completely absent. The principal importance of this work lies in the observation that the hydration dynamics of the bound water and the motion of lipid headgroups is significantly slower in the nanochannels of the Ia3d phase than the Pn3m phase and this is credited to the subtle differences in topology and other geometrical aspects. The greater negative curvature of the lipid layer, higher curvature elastic energy and more hydrophobic packing stress in the Ia3d phases is expected to restrict the flexibility of the lipid headgroups and consequently also the rotational mobility of the water molecules bound to them compared to the Pn3m phase. The different microviscosities in the two phases have been evaluated by time resolved fluorescence anisotropy of C-343 and it is apparent that the microviscosity in the Ia3d phase is higher than the Pn3m phase.


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Supporting Information The detailed experimental procedure and images for polarised light microscopy, steady state emission spectra and lifetime profiles of C-343 and C-480 in Ia3d, time resolved emission spectra of C-343 and C-480 in Ia3d phases are provided in ESI. Acknowledgement P.H. acknowledges Science and Engineering Research Board (SERB), Government of India (EMR/2016/004787) for financial support. K.D., B.R. and S.S. are thankful to IISER for providing excellent facilities and fellowship. K.D is thankful to Infosys Foundation for travel support. The authors thank anonymous reviewers for their valuable suggestions and comments. The authors would like to thank Mr. Prasad Gosavi and Mr. Roshan Dsouza of Anton Paar India Pvt. Ltd. and Dr. P. A. Hassan of Bhabha Atomic Research Centre for helping out with small-angle X-ray scattering experiments and helpful discussions.

REFERENCES 1. Pebay-Peyroula, E.; Rummel, G.; Rosenbusch, J. P.; Landau, E. M. X-ray Structure of Bacteriorhodopsin at 2.5 Angstroms from Microcrystals Grown in Lipidic Cubic Phases. Science 1997, 277, 1676. 2. Zabara, A.; Amar-Yuli, I.; Mezzenga, R. Tuning in-meso-Crystallized Lysozyme Polymorphism by Lyotropic Liquid Crystal Symmetry. Langmuir 2011, 27, 6418-6425. 3. Angelova, A.; Angelov, B.; Mutafchieva, R.; Lesieur, S.; Couvreur, P. Self-Assembled Multicompartment Liquid Crystalline Lipid Carriers for Protein, Peptide, and Nucleic Acid Drug Delivery. Acc. Chem. Res. 2011, 44, 147-156.



4. Lendermann, J.; Winter, R. Interaction of Cytochrome C with Cubic Monoolein Mesophases at Limited Hydration Conditions: The Effects of Concentration, Temperature and Pressure. Phys. Chem. Chem. Phys. 2003, 5, 1440-1450. 5. Negrini, R.; Mezzenga, R. pH-Responsive Lyotropic Liquid Crystals for Controlled Drug Delivery. Langmuir 2011, 27, 5296-5303. 6. Garti, N.; Libster, D.; Aserin, A. Lipid Polymorphism in Lyotropic Liquid Crystals for Triggered Release of Bioactives. Food Funct. 2012, 3, 700-713. 7. Mezzenga, R.; Schurtenberger, P.; Burbidge, A.; Michel, M. Understanding Foods as Soft Materials. Nat. Mater. 2005, 4, 729. 8. Kulkarni, C. V.; Wachter, W.; Iglesias-Salto, G.; Engelskirchen, S.; Ahualli, S. Monoolein: A Magic Lipid? Phys. Chem. Chem. Phys. 2011, 13, 3004-3021. 9. Qiu, H.; Caffrey, M. The Phase Diagram of the Monoolein/Water System: Metastability and Equilibrium Aspects. Biomaterials 2000, 21, 223-234. 10. Kulkarni, C. V. Lipid Crystallization: From Self-assembly to Hierarchical and Biological Ordering. Nanoscale 2012, 4, 5779-5791. 11. Mishraki-Berkowitz, T.; Ben Ishai, P.; Aserin, A.; Feldman, Y.; Garti, N. The Dielectric Study of Insulin-loaded Reverse Hexagonal (HII) Liquid Crystals. Phys. Chem. Chem. Phys. 2015, 17, 9499-9508. 12. Seddon, J. M.; Robins, J.; Gulik-Krzywicki, T.; Delacroix, H. Inverse Micellar Phases of Phospholipids and Glycolipids. Invited Lecture. Phys. Chem. Chem. Phys. 2000, 2, 4485-4493. 13. Amar-Yuli, I.; Libster, D.; Aserin, A.; Garti, N. Solubilization of Food Bioactives within Lyotropic Liquid Crystalline Mesophases. Curr. Opin. Colloid Interface Sci. 2009, 14, 21-32. 14. Larsson, K. Cubic Lipid-Water Phases: Structures and Biomembrane Aspects. J. Chem. Phys. 1989, 93, 7304-7314.


Page 26 of 33

Page 27 of 33 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


15. Fong, C.; Le, T.; Drummond, C. J. Lyotropic Liquid Crystal Engineering–ordered Nanostructured Small Molecule Amphiphile Self-assembly Materials by Design. Chem. Soc. Rev. 2012, 41, 1297-1322. 16. van ‘t Hag, L.; Gras, S. L.; Conn, C. E.; Drummond, C. J. Lyotropic Liquid Crystal Engineering Moving beyond Binary Compositional Space – Ordered Nanostructured Amphiphile Self-assembly Materials by Design. Chem. Soc. Rev. 2017, 46, 2705-2731. 17. Shah, J. C.; Sadhale, Y.; Chilukuri, D. M. Cubic Phase Gels as Drug Delivery Systems. Adv. Drug Delivery Rev. 2001, 47, 229-250. 18. Drummond, C. J.; Fong, C. Surfactant Self-assembly Objects as Novel Drug Delivery Vehicles. Curr. Opin. Colloid Interface Sci. 1999, 4, 449-456. 19. Borshchevskiy, V.; Moiseeva, E.; Kuklin, A.; Büldt, G.; Hato, M.; Gordeliy, V. Isoprenoidchained Lipid β-XylOC16+4 – A Novel Molecule for In Meso Membrane Protein Crystallization. J. Cryst. Growth 2010, 312, 3326-3330. 20. Zabara, A.; Chong, J. T. Y.; Martiel, I.; Stark, L.; Cromer, B. A.; Speziale, C.; Drummond, C. J.; Mezzenga, R. Design of Ultra-swollen Lipidic Mesophases for the Crystallization of Membrane Proteins with Large Extracellular Domains. Nat. Commun. 2018, 9, 544. 21. Vallooran, J. J.; Handschin, S.; Pillai, S. M.; Vetter, B. N.; Rusch, S.; Beck, H.-P.; Mezzenga, R. Lipidic Cubic Phases as a Versatile Platform for the Rapid Detection of Biomarkers, Viruses, Bacteria, and Parasites. Adv. Funct. Mater. 2015, 26, 181-190. 22. Zahid, N. I.; Abou-Zied, O. K.; Hashim, R. Evidence of Basic Medium in the Polar Nanochannels of the Inverse Bicontinuous Cubic Phase of a Guerbet Glycolipid: A SteadyState and Time-Resolved Fluorescence Study. J. Phys. Chem. C 2013, 117, 26636-26643. 23. Bhattacharyya, K. Solvation Dynamics and Proton Transfer in Supramolecular Assemblies. Acc. Chem. Res. 2003, 36, 95-101.



24. Pal, S. K.; Zewail, A. H. Dynamics of Water in Biological Recognition. Chem. Rev. 2004, 104, 2099-2124. 25. Roy, B.; Satpathi, S.; Gavvala, K.; Koninti, R. K.; Hazra, P. Solvation Dynamics in Different Phases of the Lyotropic Liquid Crystalline System. J. Phys. Chem. B 2015, 119, 11721-11731. 26. Wachter, W.; Trimmel, G.; Buchner, R.; Glatter, O. Dynamics of Water Confined in Selfassembled Monoglyceride–water–oil phases. Soft Matter 2011, 7, 1409-1417. 27. Ishai, P. B.; Libster, D.; Aserin, A.; Garti, N.; Feldman, Y. Molecular Interactions in Lyotropic Reverse Hexagonal Liquid Crystals: A Dielectric Spectroscopy Study. J. Phys. Chem. B 2009, 113, 12639-12647. 28. Kim, J.; Lu, W.; Qiu, W.; Wang, L.; Caffrey, M.; Zhong, D. Ultrafast Hydration Dynamics in the Lipidic Cubic Phase: Discrete Water Structures in Nanochannels. J. Phys. Chem. B 2006, 110, 21994-22000. 29. Bhattacharyya, K. Reviews in Fluorescence. Springer US: 2004. 30. Sengupta, A.; Khade, R. V.; Hazra, P. How Does the Urea Dynamics Differ from Water Dynamics inside the Reverse Micelle? J. Phys. Chem. A 2011, 115, 10398-10407. 31. Lakowicz, J. R. Principles of Fluorescence Spectroscopy. 3rd edition ed.; Springer, Boston, MA. 32. Shiraishi, Y.; Inoue, T.; Hirai, T. Local Viscosity Analysis of Triblock Copolymer Micelle with Cyanine Dyes as a Fluorescent Probe. Langmuir 2010, 26, 17505-17512. 33. Vincent, M.; Gallay, J. Water Gradient in the Membrane –Water Interface: A TimeResolved Study of the Series of n-(9-Anthroyloxy) Stearic Acids Incorporated in AOT/Water/iso-octane Reverse Micelles. J. Phys. Chem. B 2012, 116, 1687-1699.


Page 28 of 33

Page 29 of 33 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


34. Park, S.-Y.; Kwon, O.-H.; Kim, T. G.; Jang, D.-J. Ground-State Proton Transfer of 7Hydroxyquinoline Confined in Biologically Relevant Water Nanopools. J. Phys. Chem. C 2009, 113, 16110-16115. 35. Liu, X.; Cole, J. M.; Low, K. S. Solvent Effects on the UV–vis Absorption and Emission of Optoelectronic Coumarins: a Comparison of Three Empirical Solvatochromic Models. J. Phys. Chem. C 2013, 117, 14731-14741. 36. Liu, X.; Cole, J. M.; Low, K. S. Molecular Origins of Dye Aggregation and Complex Formation Effects in Coumarin 343. J. Phys. Chem. C 2013, 117, 14723-14730. 37. Correa, N. M.; Levinger, N. E. What Can You Learn from a Molecular Probe? New Insights on the Behavior of C343 in Homogeneous Solutions and AOT Reverse Micelles. J. Phys. Chem. B 2006, 110, 13050-13061. 38. Eremina, N. S.; Kopylova, T. N.; Samsonova, L. G.; Svetlichnyi, V. A. SpectralLuminescent and Lasing Properties of Aminocoumarin Derivatives in Thin Polymer Films. J. Appl. Spectrosc. 2005, 72, 499-502. 39. Lakowicz, J. R.; Keating-Nakamoto, S. Red-edge Excitation of Fluorescence and Dynamic Properties of Proteins and Membranes. Biochemistry 1984, 23, 3013-3021. 40. Chattopadhyay, A.; Haldar, S. Dynamic Insight into Protein Structure Utilizing Red Edge Excitation Shift. Acc. Chem. Res. 2014, 47, 12-19. 41. Maroncelli, M.; Fleming, G. R. Picosecond Solvation Dynamics of Coumarin 153: The Importance of Molecular Aspects of Solvation. J. Chem. Phys. 1987, 86, 6221-6239. 42. Jimenez, R.; Fleming, G. R.; Kumar, P. V.; Maroncelli, M. Femtosecond Solvation Dynamics of Water. Nature 1994, 369, 471. 43. Fee, R. S.; Maroncelli, M. Estimating the Time-zero Spectrum in Time-resolved Emmsion Measurements of Solvation Dynamics. Chem. Phys. 1994, 183, 235-247.



44. Shirota, H.; Tamoto, Y.; Segawa, H. Dynamic Fluorescence Probing of the Microenvironment of Sodium Dodecyl Sulfate Micelle Solutions: Surfactant Concentration Dependence and Solvent Isotope Effect. J. Phys. Chem. A 2004, 108, 3244-3252. 45. Biswas, S.; Santra, S.; Yesylevskyy, S.; Maiti, J.; Jana, M.; Das, R. Picosecond Solvation Dynamics in Nanoconfinement: Role of Water and Host–Guest Complexation. J. Phys. Chem. B 2018, 122, 3996-4005. 46. Bellissent-Funel, M. C.; Teixeira, J. Dynamics of Water Studied by Coherent and Incoherent Inelastic Neutron Scattering. J. Mol. Struct. 1991, 250, 213-230. 47. George, D. K.; Charkhesht, A.; Hull, O. A.; Mishra, A.; Capelluto, D. G. S.; Mitchell-Koch, K. R.; Vinh, N. Q. New Insights into the Dynamics of Zwitterionic Micelles and Their Hydration Waters by Gigahertz-to-Terahertz Dielectric Spectroscopy. J. Phys. Chem. B 2016, 120, 10757-10767. 48. Bellissent-Funel, M. C.; Chen, S. H.; Zanotti, J. M. Single-particle Dynamics of Water Molecules in Confined Space. Phys. Rev. E 1995, 51, 4558-4569. 49. Srivastava, A.; Debnath, A. Hydration Dynamics of a Lipid Membrane: Hydrogen Bond Networks and Lipid-lipid Associations. J. Chem. Phys. 2018, 148, 094901. 50. Bhattacharyya, K.; Bagchi, B. On the Origin of the Anomalous Ultraslow Solvation Dynamics in Heterogeneous Environments. J. Chem. Sci. 2007, 119, 113-121. 51. Chakrabarty, D.; Seth, D.; Chakraborty, A.; Sarkar, N. Dynamics of Solvation and Rotational Relaxation of Coumarin 153 in Ionic Liquid Confined Nanometer-Sized Microemulsions. J. Phys. Chem. B 2005, 109, 5753-5758. 52. Hara, K.; Kuwabara, H.; Kajimoto, O. Pressure Effect on Solvation Dynamics in Micellar Environment. J. Phys. Chem. A 2001, 105, 7174-7179. 53. Nandi, N.; Bagchi, B. Dielectric Relaxation of Biological Water. J. Phys. Chem. B 1997, 101, 10954-10961.


Page 30 of 33

Page 31 of 33 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


54. Nandi, N.; Bagchi, B. Anomalous Dielectric Relaxation of Aqueous Protein Solutions. J. Phys. Chem. A 1998, 102, 8217-8221. 55. Cassol, R.; Ge, M.-T.; Ferrarini, A.; Freed, J. H. Chain Dynamics and the Simulation of Electron Spin Resonance Spectra from Oriented Phospholipid Membranes. J. Phys. Chem. B 1997, 101, 8782-8789. 56. Mackay, A. L. Periodic Minimal Surfaces. Nature 1985, 314, 604. 57. Sun, W.; Vallooran, J. J.; Mezzenga, R. Enzyme Kinetics in Liquid Crystalline Mesophases: Size Matters, But Also Topology. Langmuir 2015, 31, 4558-4565. 58. Assenza, S.; Mezzenga, R. Curvature and Bottlenecks Control Molecular Transport in Inverse Bicontinuous Cubic Phases. J. Chem. Phys. 2018, 148, 054902. 59. Ghanbari, R.; Assenza, S.; Mezzenga, R. The Interplay of Channel Geometry and Molecular Features Determines Diffusion in Lipidic Cubic Phases. J. Chem. Phys. 2019, 150, 094901. 60. Shearman, G. C.; Ces, O.; Templer, R. H.; Seddon, J. M. Inverse Lyotropic Phases of Lipids and Membrane Curvature. J. Phys.: Condens. Matter 2006, 18, S1105. 61. Shearman, G. C.; Khoo, B. J.; Motherwell, M.-L.; Brakke, K. A.; Ces, O.; Conn, C. E.; Seddon, J. M.; Templer, R. H. Calculations of and Evidence for Chain Packing Stress in Inverse Lyotropic Bicontinuous Cubic Phases. Langmuir 2007, 23, 7276-7285. 62. Kulkarni, C. V.; Tang, T.-Y.; Seddon, A. M.; Seddon, J. M.; Ces, O.; Templer, R. H. Engineering Bicontinuous Cubic Structures at the Nanoscale - The Role of Chain Splay. Soft Matter 2010, 6, 3191-3194. 63. Roy, S.; Skoff, D.; Perroni, D. V.; Mondal, J.; Yethiraj, A.; Mahanthappa, M. K.; Zanni, M. T.; Skinner, J. L. Water Dynamics in Gyroid Phases of Self-Assembled Gemini Surfactants. J. Am. Chem. Soc. 2016, 138, 2472-2475.



64. Fleming, G. R. Chemical Applications of Ultrafast Spectroscopy. Oxford University Press: New York, 1986. 65. Tamai, N.; Ishikawa, M.; Kitamura, N.; Masuhara, H. Microviscosity in Polyacrylamide Gels with Pendant Triphenyl-Methane Leuco Derivatives: Picosecond Time-Resolved Fluorescence Study. Chem. Phys. Lett. 1991, 184, 398-403.


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Table of Contents

Hydration dynamics of C-480 in the Ia3d and Pn3m phases.


Impact of Topology on the Characteristics of Water inside Cubic

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