Direct Visualisation of the Structural Transformation between the

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Biophysical Chemistry, Biomolecules, and Biomaterials; Surfactants and Membranes

Direct Visualisation of the Structural Transformation between the Lyotropic Liquid Crystalline Lamellar and Bicontinuous Cubic Mesophase Nhiem Tran, Jiali Zhai, Charlotte E Conn, Xavier Mulet, Lynne J Waddington, and Calum J. Drummond J. Phys. Chem. Lett., Just Accepted Manuscript • Publication Date (Web): 29 May 2018 Downloaded from http://pubs.acs.org on May 29, 2018

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The Journal of Physical Chemistry Letters

Direct Visualisation of the Structural Transformation between the Lyotropic Liquid Crystalline Lamellar and Bicontinuous Cubic Mesophase

Nhiem Trana,

b, c, 1, *

, Jiali Zhaia,

b, 1

, Charlotte E. Conna, 1, Xavier Muletb, Lynne J.

Waddingtonb, Calum J. Drummonda, * a

School of Science, RMIT University, Melbourne, Victoria, Australia

b

CSIRO Manufacturing, Clayton, Victoria, Australia

c

Australian Synchrotron, ANSTO, Clayton, Victoria, Australia

1

These authors contributed equally

* Corresponding authors: Dr Nhiem Tran School of Science, RMIT University GPO Box 2476, Melbourne, Vic 3000 Ph: +61 3 9925 2131 Email:[email protected]

Professor Calum J. Drummond Email: [email protected]

ACS Paragon Plus Environment

The Journal of Physical Chemistry Letters 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 The transition between the lyotropic liquid crystalline lamellar and the bicontinuous cubic mesophase drives multiple fundamental cellular processes involving changes in cell membrane topology including endocytosis and membrane budding. While several theoretical models have been proposed to explain this dynamic transformation, experimental validation of these models has been challenging due to the short lived nature of the intermediates present during the phase transition. Herein, we report the direct observation of a lamellar to bicontinuous cubic phase transition in nanoscale dispersions using a combination of cryogenic transmission electron microscopy and static small angle X-ray scattering. The results represent the first experimental confirmation of a theoretical model which proposed that the bicontinuous cubic phase originates from the centre of a lamellar vesicle, then propagates outward via the formation of inter-lamellar attachments and stalks. The observation was possible due to the precise control of the lipid composition to place the dispersion systems at the phase boundary of a lamellar and a cubic phase, allowing for the creation of long-lived structural intermediates. By surveying the nanoparticles using cryogenic transmission electron microscopy, a complete phase transition sequence was established. Table of Contents Graphic


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The amphiphilic nature of lipid molecules drives them to adopt complex nanostructures in the presence of water as a result of the hydrophobic effect. The most ubiquitous of these phases biologically is the lamellar phase, in which the lipid molecules self-assemble into flat sheets of lipid bilayers, and which forms the fundamental structural motif of all cell membranes. Under certain conditions of pressure, temperature, or composition, instead of forming flat sheets, the lipid molecules assemble into negatively curved surfaces such as the inverse bicontinuous cubic phases.1 These unique nanostructures can be described as a bilayer draped along an infinitely periodic minimal surface, and dividing space into two noninterconnected aqueous volumes.2 The inverse bicontinuous cubic phases are believed to act as structural intermediates in many dynamic cellular processes that involve changes in membrane topology, including endo- and exocytosis, fat digestion, and membrane budding.3-4 Additionally, their complex nanostructures and biocompatibility make them suitable for the delivery of a wide variety of therapeutics including small molecule drugs, proteins, peptides and nucleic-acid based therapeutics.5-7 Although much is understood about the formation of the mesophases in equilibrium, the mechanistic pathways that describe the structural transitions between these phases have received considerably less attention, with even fewer studies focussing on the confined environment of a nanoparticle. Theoretical models of the structural transition between a lamellar and a cubic phase are generally based on the formation of “stalks” between apposed lipid bilayers.8-9 Experimental attempts to provide evidence for the stalk theory of membrane fusion have relied on temperature or pressure-jumps to initiate the transition between the lamellar and cubic phases.10-17 Evidence for the role of structural intermediates based on stalks has been observed in some studies. 10, 15, 18 However, another study showed that the vesicle-cubosome transition appeared to proceed directly, with no, or only minor, evidence of structural



intermediates observed 19. Conn et al. studied the lamellar to cubic transition within vesicles using pressure jumps and proposed a qualitative model to explain the mechanism of the transition (Figure 1). They proposed that thermal fluctuations between concentric bilayers (Figure 1a) initially result in the formation of inter-lamellar attachments (ILAs) or stalks between the bilayers (Figure 1b). The lateral tension exerted by these attachments will eventually lead to rupture and the formation of a disordered array of fusion pores (Figure 1c). They also argued that a localised cubic phase, located at the centre of the onion vesicle, acts as a seed for the growth of the bicontinuous cubic phase. The bilayer can bend in order to equalise curvature over the surface, resulting in the formation of the symmetric bicontinuous cubic phase (Figure 1d). Some studies have provided visual cryo-TEM images of the later stages of the lamellar – cubic transition, including inter-lamellar attachments and fusion pores.10,

12, 14, 20

However, a key feature of the proposed transition pathway is the role of

centralised bicontinuous cubic phase, which to our knowledge has not been observed via cryo-TEM.


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Figure 1. The proposed structural rearrangements during the lamellar to bicontinuous cubic phase transition. Reproduced with permission from 15. Difficulties in studying phase transitions experimentally mainly reflect technical challenges when monitoring dynamic transitions between liquid crystalline phases. In general, transitions involving no change in topology, such as between the cubic phases, are extremely fast, occurring within milliseconds.21 Transitions which involve significant changes in topology, including from the lamellar to cubic phase, are generally slower, but can still occur on a timescale of less than a minute. Several studies have utilised the powerful synchrotron light source and time-resolved small angle X-ray scattering (TR-SAXS) technique to monitor these rapid structural changes. 11, 13-14 A different approach is to create long-living intermediates by altering the composition of the lipid mixtures. This technique, sometime termed “compositional melting”, was initially used to adjust the melting temperature of a lipid by adding in another amphiphile.13 Mulet et al. found that addition of the block copolymer Poloxamer 407 (also called Pluronic F127) to a prodrug (5’-deoxy-5fluoro-N4-(cis-9-octadecenyl-oxycarbonyl) cytidine) resulted in a long-living metastable intermediate. This phase “X” was observed during a temperature jump experiment and was proposed as the precursor of a cubic phase.10 Herein, we have used the compositional melting technique, with the aid of SAXS and cryo-TEM, to observe the structural intermediates during the lamellar to cubic transition in nanoparticles, which provide the visual evidence for the model that was proposed by Conn et al.15 By carefully adjusting the lipid composition, we were able to prepare nanoparticles with mesophases at the boundary of cubic and lamellar phase. At this composition, when surveying the sample using cryo-TEM, we found a variety of nanoparticles, some of which are cubic, some are lamellar, and some are intermediates of different stages between lamellar and cubic phase. The visual analysis of these stable



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nanoparticles allows us to directly visualise the sequential events which occur during the lamellar-cubic transition, without the need of temperature or pressure jumps. The system of interest consisted of cubic phase forming lipid monoolein (MO), capric acid (CA), and 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC). The nanoparticles were stabilised by a block copolymer, Pluronic F127. Similar to previous works, the nanoparticles formed appeared to be homogenous with no visual phase separation for days.22-23 In our previous study, we demonstrated that this ternary system allowed us to precisely control the formation of a variety of mesophases including lamellar, cubic, and hexagonal phase.24 In the current study, the weight ratio of CA to MO and the weight ratio of F127 to MO were fixed at 0.25 and 0.1, respectively. The pH of the aqueous phase was 4.8. At this pH, CA exists in the protonated form.24-25 The surface curvature and mesophase were controlled by adjusting the amount of DLPC in the mixture, which can be described by =

( )

. The

( )

cylindrical architecture of the DLPC molecule drives a reduction in interfacial curvature. An increase in α therefore promotes the formation of structures with less curved interfaces such as the lamellar phase. Conversely, a decrease in α drives the system toward mesophases with more negatively curved surfaces such as the bicontinuous cubic and hexagonal phases. At α = 0.4, a broad peak was observed in the synchrotron SAXS pattern at a q-value of approximately 0.15 Å-1, consistent with the formation of multi-membrane vesicles (Figure 2a). The formation of such vesicles were clearly visualised via cryo-TEM in Figure 2b. The absence of Bragg peaks corresponding to a lamellar phase in the SAXS pattern indicates that the lamellar layers are quite loosely packed. When α was reduced to 0.3, several scattering peaks are observed superimposed on the broad background peak in the SAXS profile of the nanoparticles, indicating the coexistence of ordered structures with the multi-membrane vesicles (Figure 2c). The identifiable peaks with spacing ratios of √2: √4: √6: √14 were assigned to be the (110), (200), (211) and (321) reflections of a primitive cubic phase, QIIP


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(space group Im3m) (Figure 2d). This cubic phase with a calculated lattice parameter of 135 Å is typically for MO cubosomes stabilised with F127. 22, 26

Figure 2. (a) SAXS profile of multi membrane vesicles of MO-CA- DPLC at α = 0.4, where α represents the ratio of the amount of DLPC to the total amount of the ternary lipid mixtures in weight. (b) cryo-TEM of multi membrane vesicles whose SAXS profile presented in (a). (c) SAXS profile of a MO-CA- DPLC dispersion at α = 0.3, showing the unique scattering peaks of a primitive (QIIP) cubic phase over a broad scattering of multi membrane vesicles. (d) The peaks are indexed using standard reflections of primitive (Im3m). q(hkl) was plotted against d-spacing ratio (h2+k2+l2)1/2, where h, k, and l are Millers indices. The SAXS profile shows several peaks with spacing ratios of √2: √4: √6 : √14. They can be indexed as the (110), (200), (211) and (321) reflections of a primitive (QIIP, space group Im3m) cubic phase respectively. Cryo-TEM analysis confirmed the coexistence of lamellar and cubic phases in samples with α = 0.3. A series of cryo-TEM images displaying a range of nanoparticle morphologies representing the proposed structural transition pathway from the lamellar to the cubic phase is presented in Figure 3. Figure 3a shows the concentric bilayers of an “onion-like” multi membrane vesicle. Towards the centre of the vesicle, where the bilayer curvature is extremely high, a more disordered structure is observed. Inter-lamellar attachments (ILAs)



between the bilayers may be clearly seen in the particle displayed in Figure 3b. In Figure 3c, rupture of the ILAs can be seen, with the subsequent formation of a nanoparticle morphology consisting of a disordered network of pores (Figure 3d). The formation of this porous internal network allows for the diffusion of water from the centre of the vesicle and large water storage depots may indeed now be observed just within the external bilayer envelope of the nanoparticle in Figure 3d. Relaxation of this disordered network results in increasingly wellordered cubosomes, displaying the characteristic internal cubic periodicity (Figure 3e and f, respectively). In Figure 3e, localisation of the cubic phase to the centre of the particle may be clearly observed, whereas in Figure 3f the cubic phase has expanded to fill the particle. In this study, a total of 212 lipid nanoparticles were assessed by cryo-TEM, and 196 of which were categorised as falling within one of the six stages shown in Figure 3 (Figure S1). To demonstrate the reproducibility of this sequence, a similar series of particles morphologies is provided in the Supporting Information Figure S2.


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Figure 3. Nanoparticle morphologies as observed via cryo-TEM in a MO-CA-DLPC dispersion at the boundary of lamellar-cubic phase. The images are arranged to depict the transition from lamellar to bicontinuous cubic phase. Arrows show selected points where membranes detach and inter-lamellar attachments (ILA) are formed. Inset of (e) and (f) show fast Fourier transformation of the internal cubic phase. Scale bars = 100 nm. Arrows The observed morphologies provide, for the first time, direct visual evidence for the theoretical structural pathway as proposed in Conn et al. and reproduced in Figure 1.15 In Figure 3, it can be seen that the bicontinuous cubic phase originated from the centre of the multi membrane vesicle and spread out gradually. It was suggested that the existence of the central, highly swollen cubic phase would both act to relieve unfavourably high interfacial curvature towards the centre of the onion vesicle and potentially act as a water storage depot. Increasing number of ILAs observed in Figure 3b and Figure 3c is associated with an increase in interfacial tension; the eventual rupture of these ILAs then gives rise to the porous internal network observed in Figure 3d. In our particular case, the transition was between lamellar and primitive cubic phase (QIIP), which is only one of three bicontinuous cubic structures based on Schwarz minimal surfaces. It would be interesting to see whether the transitions between lamellar and the other two cubic phases (i.e. diamond (Pn3m) and gyroid (Ia3d) cubic phases) would occur via similar intermediates. This is likely because these three cubic phases are almost isoenergetic. In any case, the compositional melting method used here should be useful to study such lamellar – cubic transitions or the transition between other mesophases such as lamellar – hexagonal or hexagonal – cubic.

Experimental section Materials Monoolein (MO) was obtained from Nu-check-Prep, Inc. with purity >99% as determined by gas liquid chromatography. Capric acid (CA), Pluronic F127, ethanol, and chloroform were purchased from Sigma-Aldrich. 1,2-dilauroyl-sn-glycero-3-phosphocholine



(DLPC) was purchased from Avanti Lipids. Milli-Q H2O (18.2 MΩ) was used for all aqueous preparations. All compounds were used without further purification.

Nanoparticle preparation MO and CA stock solutions were dissolved in ethanol. DLPC stock solution was dissolved in chloroform. The organic solvents were then removed overnight using a centrifugal evaporator (GeneVac). The weight ratio of CA to MO was kept at 0.25. The amount of DLPC added to the ternary lipid nanoparticulate systems was defined by α:

=

Amount of PL Amount of (MO + CA + PL)

Based on our previous study on the phase behaviour of MO-CA-phospholipid system,24 samples with α = 0.4 and 0.3 were selected to ensure that the nanoparticles were either multi membrane vesicles or a mixture of cubic phase nanoparticles and multi membrane vesicles. Pluronic F127 in Milli-Q water was added to the dried lipid mixtures at 10 wt% to the total amount of bulk lipids. The samples were then sonicated by a probe sonicator (Qsonica, 30% amplitude, 5 second-on, 5 second-off, 3 minute duration).

Synchrotron small angle X-ray scattering (SAXS) Synchrotron SAXS experiment were performed at the SAXS/wide-angle X-ray scattering (WAXS) beamline at the Australian Synchrotron at 25 °C. The X-ray had a beam with a wavelength of 1.033 Å (12.0 keV) with a typical flux of approximately 1013 photons/s. The sample to detector distance was chosen as 1.6 m which provided a q-range of 0.01- 0.5 Å-1 (scattering vector q = 4π sin(θ)/λ where θ is the scattering angle and λ is the wavelength). Two dimensional X-ray diffraction images were recorded on a Decris-Pilatus 1-M detector using an in-house IDL-based ScatterBrain software.27 The scattering images were integrated into one dimensional plots of intensity versus q for phase identification. A silver behenate standard (d = 58.38 Å was used for calibration). The exposure time for each sample was 1 s.


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Prepared samples were loaded in a 96-well, half-area UV-clear plate (Greiner Bio-One) and positioned in the high-throughput plate holder at the beamline with temperatures controlled via a recirculating water bath as previously described.22 SAXS data were analysed using the IDL-based AXcess software package to determine the identity and the lattice parameter (LP) of the internal lyotropic liquid crystalline mesophase. Nanoparticle mesophases were identified using the spacing ratios of peaks in the one dimensional plot.23, 28

Cryogenic transmission electron microscopy (cryo-TEM) Cryo-TEM was used to visualize the formulated nanoparticles. Copper grids (200mesh) coated with perforated carbon film (Lacey carbon film, ProSci Tech, Australia) were glow discharged in nitrogen to render them hydrophilic and placed in a laboratory-built humidity-controlled vitrification system. Aliquots of samples were applied onto the grids and after 30 s adsorption time, grids were blotted manually by filter paper for approximately 3 s. Grids were then plunged into liquid ethane cooled by liquid nitrogen. The samples were examined using a Gatan 626 cryoholder (Gatan, Pleasanton, CA, USA) and Tecnai 12 Transmission Electron Microscope (FEI, Eindhoven, The Netherlands) at an operating voltage of 120KV. At all times low dose procedures were followed, using an electron dose of 8-10 electrons Å-2 for all imaging. Images were recorded using a FEI Eagle 4k x 4k CCD camera at magnifications ranging from 15000x to 50000x. In total, 212 nanoparticles were examined by ImageJ.

Supporting information Analysis and categorisation of nanoparticle structure by using cryo-TEM. Additional cryoTEM images describing the transition from the lamellar to the bicontinuous cubic mesophase.

Acknowledgement



N.T. acknowledges the funding from the RMIT University Vice-Chancellor’s Research Fellowship and the Science and Industry Endowment Fund (SIEF) postdoctoral fellowship. C.E.C is supported by RMIT University Vice-Chancellor’s Senior Research Fellowship and ARC DECRA. This research includes work undertaken on the SAXS/WAXS beamline at the Australian Synchrotron, ANSTO, Victoria, Australia. The authors thank the beamline scientists, Dr. Nigel Kirby, Dr. Adrian Hawley, and Dr. Stephen Mudie at the SAXS/WAXS beamline for assisting with the SAXS experiments.

Conflict of interest The authors declare no conflict of interest.


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20. Efrat, R.; Kesselman, E.; Aserin, A.; Garti, N.; Danino, D. Solubilization of hydrophobic guest molecules in the monoolein discontinuous QL cubic mesophase and its soft nanoparticles. Langmuir 2008, 25 (3), 1316-1326. 21. Conn, C. E.; Ces, O.; Squires, A. M.; Mulet, X.; Winter, R.; Finet, S. M.; Templer, R. H.; Seddon, J. M. A pressure-jump time-resolved x-ray diffraction study of cubic-cubic transition kinetics in monoolein. Langmuir 2008, 24 (6), 2331-2340. 22. Tran, N.; Hawley, A.; Zhai, J.; Muir, B. W.; Fong, C.; Drummond, C. J.; Mulet, X. Highthroughput screening of saturated fatty acid influence on nanostructure of lyotropic liquid crystalline lipid nanoparticles. Langmuir 2016, 32 (18), 4509-4520. 23. Tran, N.; Mulet, X.; Hawley, A. M.; Hinton, T. M.; Mudie, S. T.; Muir, B. W.; Giakoumatos, E. C.; Waddington, L. J.; Kirby, N. M.; Drummond, C. J. Nanostructure and Cytotoxicity of SelfAssembled Monoolein-Capric Acid Lyotropic Liquid Crystalline Nanoparticles. RSC Advances 2015, 5 (34), 26785-26795. 24. Zhai, J.; Tran, N.; Sarkar, S.; Fong, C.; Mulet, X.; Drummond, C. J. Self-assembled Lyotropic Liquid Crystalline Phase Behavior of Monoolein–Capric Acid–Phospholipid Nanoparticulate Systems. Langmuir 2017, 33 (10), 2571-2580. 25. Drummond, C. J.; Grieser, F.; Healy, T. W. Acid–base equilibria in aqueous micellar solutions. Part 1.—‘simple’weak acids and bases. Journal of the Chemical Society, Faraday Transactions 1: Physical Chemistry in Condensed Phases 1989, 85 (3), 521-535. 26. Tran, N.; Mulet, X.; Hawley, A.; Conn, C. E.; Zhai, J.; Waddington, L. J.; Drummond, C. J. First Direct Observation of Stable Internally Ordered Janus Nanoparticles Created by Lipid SelfAssembly. Nano letters 2015, 15 (6), 4229-4233. 27. Kirby, N. M.; Mudie, S. T.; Hawley, A. M.; Cookson, D. J.; Mertens, H. D.; Cowieson, N.; Samardzic-Boban, V. A low-background-intensity focusing small-angle X-ray scattering undulator beamline. Journal of Applied Crystallography 2013, 46 (6), 1670-1680. 28. Kulkarni, C. V.; Wachter, W.; Iglesias-Salto, G.; Engelskirchen, S.; Ahualli, S. Monoolein: a magic lipid? Physical Chemistry Chemical Physics 2011, 13 (8), 3004-3021.


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Direct Visualisation of the Structural Transformation between the

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