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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 because of 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 center of a lamellar vesicle then propagates outward via the formation of interlamellar attachments and stalks. The observation was possible because of 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 the surveying of the nanoparticles using cryogenic transmission electron microscopy, a complete phase transition sequence was established.

Amyloid nanofibrils are ubiquitous biological protein fibrous aggregates, with a wide range of either toxic or beneficial activities that are relevant to human disease and normal biology. Protein amyloid fibrillization occurs via nucleated polymerization, through non-covalent interactions. As such, protein nanofibril formation is based on a complex interplay between kinetic and thermodynamic factors. The process entails metastable oligomeric species and a highly thermodynamically favoured end state. The kinetics, and the reaction pathway itself, can be influenced by third party moieties, either molecules or surfaces. Specifically, in the biological context, different classes of biomolecules are known to act as catalysts, inhibitors or modifiers of the generic protein fibrillization process. The biological aggregation modifiers reviewed here include lipid membranes of varying composition, glycosaminoglycans and metal ions, with a final word on xenobiotic compounds. The corresponding molecular interactions are critically analysed and placed in the context of the mechanisms of cytotoxicity of the amyloids involved in diverse pathologies and the non-toxicity of functional amyloids (at least towards their biological host). Finally, the utilization of this knowledge towards the design of bio-inspired and biocompatible nanomaterials is explored., Competing Interests: The authors declare no competing interests.

Ordered amphiphile self-assembly materials with a tunable three-dimensional (3D) nanostructure are of fundamental interest, and crucial for progressing several biological and biomedical applications, including in meso membrane protein crystallization, as drug and medical contrast agent delivery vehicles, and as biosensors and biofuel cells. In binary systems consisting of an amphiphile and a solvent, the ability to tune the 3D cubic phase nanostructure, lipid bilayer properties and the lipid mesophase is limited. A move beyond the binary compositional space is therefore required for efficient engineering of the required material properties. In this critical review, the phase transitions upon encapsulation of more than 130 amphiphilic and soluble additives into the bicontinuous lipidic cubic phase under excess hydration are summarized. The data are interpreted using geometric considerations, interfacial curvature, electrostatic interactions, partition coefficients and miscibility of the alkyl chains. The obtained lyotropic liquid crystal engineering design rules can be used to enhance the formulation of self-assembly materials and provides a large library of these materials for use in biomedical applications (242 references).

The inverse bicontinuous lipidic cubic phase offers a simple and robust membrane mimetic with the ability to encapsulate peptides, potentially increasing bioavailability, while also offering a platform from which functionalized, targeted nanoparticles can be developed. Herein we have investigated the use of a number of cubic phase nanoparticle systems with encapsulated antimicrobial peptides gramicidin A', melittin, and alamethicin. The optimal peptide loading ranges, over which cubic symmetry was retained, were determined using small angle X-ray scattering. A large variation in peptide loading capability of different cubosome formulations was confirmed using circular dichroism. Observations are supported by particle sizing using dynamic light scattering as well as by direct visualization of nanoparticle morphology using cryogenic transmission electron microscopy. The results are discussed in relation to bilayer properties such as the hydrophobic mismatch between bilayer and peptide, intrinsic surface curvature, and lateral pressure profile of each lipid system. The findings of this study should be of use in the further development of lipid-based peptide encapsulation systems, particularly in the field of drug delivery., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The lyotropic phase behaviour of a library of sugar-based amphiphiles was investigated using high-throughput small-angle X-ray scattering (SAXS). Double unsaturated-chain monosaccharide amphiphiles formed inverse hexagonal and cubic micellar (Fd3m) lyotropic phases under excess water conditions. A galactose-oleyl amphiphile from the library was subsequently formulated into hexosome nanoparticles, which have potential uses as drug delivery vehicles. The nanoparticles were shown to be stable at elevated temperatures and non-cytotoxic up to at least 200μgmL -1 ., (Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.)

The ordered nanostructured lipidic bicontinuous cubic phase has demonstrated potential as a drug release material, due to its ability to encapsulate a wide variety of compounds, which may undergo sustained, diffusion controlled release over time. Control of drug release has been shown to depend on the nanostructural parameters of the lipid mesophase. Herein, the diffusion and release of two amino acids, encapsulated within a range of different lipidic cubic mesophases are investigated. Pulsed-field gradient NMR was used to determine the diffusion coefficient of the encapsulated amino acid, which was found to be correlated with the nanoscale diameter of the water channels within the cubic mesophase. This information was used to predict the release profiles of encapsulated compounds from within the cubic mesophase, which was verified by directly measuring the release of each amino acid in vitro. Predicted release profiles tracked reasonably close to the measured release profiles, indicating that NMR determined diffusion measurements can be used to predict release profiles.

Retention of amphiphilic protein activity within the lipid bilayer membrane of the nanostructured biomimetic bicontinuous cubic phase is crucial for applications utilizing these hybrid protein-lipid self-assembly materials, such as in meso membrane protein crystallization and drug delivery. Previous work, mainly on soluble and membrane-associated enzymes, has shown that enzyme activity may be modified when immobilized, including membrane bound enzymes. The effect on activity may be even greater for amphiphilic enzymes with a large hydrophilic domain, such as the Neisserial enzyme lipid A phosphoethanolamine transferase (EptA). Encapsulation within the biomimetic but non-endogenous lipid bilayer membrane environment may modify the enzyme conformation, while confinement of the large hydrophilic domain with the nanoscale water channels of a continuous lipid bilayer structure may prevent full function of this enzyme. Herein we show that NmEptA remains active despite encapsulation within a nanostructured bicontinuous cubic phase. Full transfer of the phosphoethanolamine (PEA) group from a 1,2-dioleoyl-glycero-phosphoethanolamine (DOPE) doped lipid to monoolein (MO), which makes up the bicontinuous cubic phase, is shown. The reaction was found to be non-specific to the alkyl chain identity. The observed rate of enzyme activity is similar to other membrane bound enzymes, with complete transfer of the PEA group occurring in vitro, under the conditions studied, over a 24 hour timescale.

The structural changes occurring at the nanoscale level within the lipid bilayer and driving the in-meso formation of large well-diffracting membrane protein crystals have been uniquely characterized for a model membrane protein, intimin. Importantly, the order to order transitions taking place within the bilayer and the lipidic nanostructures required for crystal growth have been shown to be general, occurring for both the cubic and the sponge mesophase crystallization pathways. For the first time, a transient fluid lamellar phase has been observed and unambiguously assigned for both crystallization pathways, present at the earliest stages of protein crystallogenesis but no longer observed once the crystals surpass the size of the average lyotropic liquid crystalline domain. The reported time-resolved structural investigation provides a significantly improved and general understanding of the nanostructural changes taking place within the mesophase during in-meso crystallization which is a fundamental advance in the enabling area of membrane protein structural biology.

A fundamental understanding of the effect of amphiphilic protein encapsulation on the nanostructure of the bicontinuous cubic phase is crucial to progressing biomedical and biological applications of these hybrid protein-lipid materials, including as drug delivery vehicles, as biosensors, biofuel cells and for in meso crystallization. The relationship between the lipid nanomaterial and the encapsulated protein, however, remains poorly understood. In this study, we investigated the effect of incorporating the five transmembrane and lipo-proteins which make up the β-barrel assembly machinery from Gram-negative bacteria within a series of bicontinuous cubic phases. The transmembrane β-barrel BamA caused an increase in lattice parameter of the cubic phase upon encapsulation. By contrast, the mainly hydrophilic lipo-proteins BamB-E caused the cubic phase lattice parameters to decrease, despite their large size relative to the diameter of the cubic phase water channels. Analysis of the primary amino acid sequence was used to rationalize this effect, based on specific interactions between aromatic amino acids within the proteins and the polar-apolar interface. Other factors that were found to have an effect were lateral bilayer pressure and rigidity within the lipid bilayer, water channel diameter, and size and structure of the lipo-proteins. The data presented suggest that hydrophilic bioactive molecules can be selectively encapsulated within the cubic phase by using a lipid anchor or aromatic amino acids, for drug delivery or biosensing applications.

Milk has been used commercially as a carrier for phytosterols, but there is limited knowledge on the effect of added plant sterols on the properties of the system. In this study, phytosterols dispersed in milk fat at a level of 0.3 or 0.6% were homogenized with an aqueous dispersion of whey protein isolate (WPI). The particle size, morphology, ζ-potential, and stability of the emulsions were investigated. Emulsion crystallization properties were examined through the use of differential scanning calorimetry (DSC) and Synchrotron X-ray scattering at both small and wide angles. Phytosterol enrichment influenced the particle size and physical appearance of the emulsion droplets, but did not affect the stability or charge of the dispersed particles. DSC data demonstrated that, at the higher level of phytosterol addition, crystallization of milk fat was delayed, whereas, at the lower level, phytosterol enrichment induced nucleation and emulsion crystallization. These differences were attributed to the formation of separate phytosterol crystals within the emulsions at the high phytosterol concentration, as characterized by Synchrotron X-ray measurements. X-ray scattering patterns demonstrated the ability of the phytosterol to integrate within the milk fat triacylglycerol matrix, with a concomitant increase in longitudinal packing and system disorder. Understanding the consequences of adding phytosterols, on the physical and crystalline behavior of emulsions may enable the functional food industry to design more physically and chemically stable products.

The proposed mechanism for in meso crystallization of transmembrane proteins suggests that a protein or peptide is initially uniformly dispersed in the lipid self-assembly cubic phase but that crystals grow from a local lamellar phase, which acts as a conduit between the crystal and the bulk cubic phase. However, there is very limited experimental evidence for this theory. We have developed protocols to investigate the lipid mesophase microenvironment during crystal growth using standard procedures readily available in crystallography laboratories. This technique was used to characterize the microenvironment during crystal growth of the DAP12-TM peptide using synchrotron small angle X-ray scattering (SAXS) with a micro-sized X-ray beam. Crystal growth was found to occur from the gyroid cubic mesophase. For one in four crystals, a highly oriented local lamellar phase was observed, providing supporting evidence for the proposed mechanism for in meso crystallization. A new observation of this study was that we can differentiate diffraction peaks from crystals grown in meso, from peaks originating from the surrounding lipid matrix, potentially opening up the possibility of high-throughput SAXS analysis of in meso grown crystals.This article is part of the themed issue 'Soft interfacial materials: from fundamentals to formulation'., (© 2016 The Author(s).)

An understanding of the location of peptides, proteins, and other biomolecules within the bicontinuous cubic phase is crucial for understanding and evolving biological and biomedical applications of these hybrid biomolecule-lipid materials, including during in meso crystallization and drug delivery. While theoretical modeling has indicated that proteins and additive lipids might phase separate locally and adopt a preferred location in the cubic phase, this has never been experimentally confirmed. We have demonstrated that perfectly contrast-matched cubic phases in D2O can be studied using small-angle neutron scattering by mixing fully deuterated and hydrogenated lipid at an appropriate ratio. The model transmembrane peptide WALP21 showed no preferential location in the membrane of the diamond cubic phase of phytanoyl monoethanolamide and was not incorporated in the gyroid cubic phase. While deuteration had a small effect on the phase behavior of the cubic phase forming lipids, the changes did not significantly affect our results.

Nanostructured bicontinuous lipidic cubic phases are used for the encapsulation of proteins in a range of applications such as in meso crystallization of transmembrane proteins and as drug delivery vehicles. The retention of the nanoscale order of the cubic phases subsequent to protein incorporation, as well as retention of the protein structure and function, is essential for all of these applications. Herein synthetic peptides (WALP21, WALPS53, and WALPS73) with a common α-helical hydrophobic domain, but varying hydrophilic loop size, were designed to systematically examine the effect of peptide structure and charge on bicontinuous cubic phases. The effect of the cubic phases on the secondary structure of the peptides was also investigated. The incorporation of the WALP peptides in cubic phases formed by a range of lipids showed that hydrophobic mismatch of the peptides with the lipid bilayers, the hydrophilic domain size, and peptide charge were all significant factors determining the response of the lipid nanomaterial to protein insertion. As charge repulsion had the most significant effect on the phase transitions observed, we suggest that buffer pH and salt concentration must be carefully considered to ensure cubic mesophase retention. Importantly, the WALP peptides were found to have a different conformation depending on the local lipid environment. Such structural changes could potentially affect membrane protein function, which is crucial for both current and prospective applications.

Main Conclusion: A set of PpKAI2 - LIKE paralogs that may encode strigolactone receptors in Physcomitrella patens were identified through evolutionary, structural, and transcriptional analyses, suggesting that strigolactone perception may have evolved independently in basal land plants in a similar manner as spermatophytes. Carotenoid-derived compounds known as strigolactones are a new class of plant hormones that modulate development and interactions with parasitic plants and arbuscular mycorrhizal fungi. The strigolactone receptor protein DWARF14 (D14) belongs to the α/β hydrolase family. D14 is closely related to KARRIKIN INSENSITIVE2 (KAI2), a receptor of smoke-derived germination stimulants called karrikins. Strigolactone and karrikin structures share a butenolide ring that is necessary for bioactivity. Charophyte algae and basal land plants produce strigolactones that influence their development. However phylogenetic studies suggest that D14 is absent from algae, moss, and liverwort genomes, raising the question of how these basal plants perceive strigolactones. Strigolactone perception during seed germination putatively evolved in parasitic plants through gene duplication and neofunctionalization of KAI2 paralogs. The moss Physcomitrella patens shows an increase in KAI2 gene copy number, similar to parasitic plants. In this study we investigated whether P. patens KAI2-LIKE (PpKAI2L) genes may contribute to strigolactone perception. Based on phylogenetic analyses and homology modelling, we predict that a clade of PpKAI2L proteins have enlarged ligand-binding cavities, similar to D14. We observed that some PpKAI2L genes have transcriptional responses to the synthetic strigolactone GR24 racemate or its enantiomers. These responses were influenced by light and dark conditions. Moreover, (+)-GR24 seems to be the active enantiomer that induces the transcriptional responses of PpKAI2L genes. We hypothesize that members of specific PpKAI2L clades are candidate strigolactone receptors in moss.

The α/β-hydrolases KAI2 and D14 are paralogous receptors for karrikins and strigolactones, two classes of plant growth regulators with butenolide moieties. KAI2 and D14 act in parallel signaling pathways that share a requirement for the F-box protein MAX2, but produce distinct growth responses by regulating different members of the SMAX1-LIKE/D53 family. kai2 and max2 mutants share seed germination, seedling growth, leaf shape, and petiole orientation phenotypes that are not found in d14 or SL-deficient mutants. This implies that KAI2 recognizes an unknown, endogenous signal, herein termed KAI2 ligand (KL). Recent studies of ligand-specificity among KAI2 paralogs in basal land plants and root parasitic plants suggest that karrikin and strigolactone perception may be evolutionary adaptations of KL receptors. Here we demonstrate that evolutionarily conserved KAI2c genes from two parasite species rescue multiple phenotypes of the Arabidopsis kai2 mutant, unlike karrikin-, and strigolactone-specific KAI2 paralogs. We hypothesize that KAI2c proteins recognize KL, which could be an undiscovered hormone.

Lipidic bicontinuous cubic mesophases with encapsulated amphiphilic proteins are widely used in a range of biological and biomedical applications, including in meso crystallization, as drug delivery vehicles for therapeutic proteins, and as biosensors and biofuel cells. However, the effect of amphiphilic protein encapsulation on the cubic phase nanostructure is not well-understood. In this study, we illustrate the effect of incorporating the bacterial amphiphilic membrane protein Ag43, and its individual hydrophobic β(43) and hydrophilic α(43) domains, in bicontinuous cubic mesophases. For the monoolein, monoalmitolein, and phytantriol cubic phases with and without 8% w/w cholesterol, the effect of the full length amphiphilic protein Ag43 on the cubic phase nanostructure was more significant than the sum of the individual hydrophobic β(43) and hydrophilic α(43) domains. Several factors were found to potentially influence the impact of the hydrophobic β(43) domain on the cubic phase internal nanostructure. These include the size of the hydrophobic β(43) domain relative to the thickness of the lipid bilayer, as well as its charge and diameter. The size of the hydrophilic α(43) domain relative to the water channel radius of the cubic mesophase was also found to be important. The secondary structure of the Ag43 proteins was affected by the hydrophobic thickness and physicochemical properties of the lipid bilayer and the water channel diameter of the cubic phase. Such structural changes may be small but could potentially affect membrane protein function.

Obligate parasitic plants in the Orobanchaceae germinate after sensing plant hormones, strigolactones, exuded from host roots. In Arabidopsis thaliana, the α/β-hydrolase D14 acts as a strigolactone receptor that controls shoot branching, whereas its ancestral paralog, KAI2, mediates karrikin-specific germination responses. We observed that KAI2, but not D14, is present at higher copy numbers in parasitic species than in nonparasitic relatives. KAI2 paralogs in parasites are distributed into three phylogenetic clades. The fastest-evolving clade, KAI2d, contains the majority of KAI2 paralogs. Homology models predict that the ligand-binding pockets of KAI2d resemble D14. KAI2d transgenes confer strigolactone-specific germination responses to Arabidopsis thaliana. Thus, the KAI2 paralogs D14 and KAI2d underwent convergent evolution of strigolactone recognition, respectively enabling developmental responses to strigolactones in angiosperms and host detection in parasites., (Copyright © 2015, American Association for the Advancement of Science.)

We present the first observation of Janus nanoparticles consisting of stable, coexisting ordered mesophases in discrete particles created by lipid self-assembly. Cryo-TEM images provided visual identification of the multicompartment Janus nanoparticles and, combined with SAXS data, confirmed the presence of mixed cubic phases and mixed cubic/hexagonal phases within individual nanoparticles. We further investigated computer visualization models to interpret the potential interface between the interconnected coexisting nanostructured domains within a single nanoparticle.

The membrane-spanning α helices of single-pass receptors play crucial roles in stabilizing oligomeric structures and transducing biochemical signals across the membrane. Probing intermolecular transmembrane interactions in single-pass receptors presents unique challenges, reflected in a gross underrepresentation of their membrane-embedded domains in structural databases. Here, we present two high-resolution structures of transmembrane assemblies from a eukaryotic single-pass protein crystallized in a lipidic membrane environment. Trimeric and tetrameric structures of the immunoreceptor signaling module DAP12, determined to 1.77-Å and 2.14-Å resolution, respectively, are organized by the same polar surfaces that govern intramembrane assembly with client receptors. We demonstrate that, in addition to the well-studied dimeric form, these trimeric and tetrameric structures are made in cells, and their formation is competitive with receptor association in the ER. The polar transmembrane sequences therefore act as primary determinants of oligomerization specificity through interplay between charge shielding and sequestration of polar surfaces within helix interfaces., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

The butyrate G-protein coupled receptors (GPCRs), GPR41 and GPR43, have been implicated in colorectal cancer and leptin production. To date their function has not been elucidated as low levels of protein expression and difficulties in producing diffraction quality crystals have hindered their structural determination. In meso crystallization, which uses an artificial lipid membrane matrix to facilitate crystal growth, is becoming an increasingly successful crystallization technique, particularly for GPCRs. We report herein the lipid membrane matrix structural characterization for GPR41 and GPR43 within two lipid self-assembly systems (monoolein and phytantriol) commonly used for in meso crystallization and comment on their suitability for crystallizing these GPCRs. Synchrotron small angle X-ray scattering (SAXS) studies were used to determine the initial phase and uptake of these receptors within the lipid matrix and investigate the role of cholesterol in this process. The self-assembled lipid nanostructure was retained in the presence of GPR43 for both lipids but was destabilized for GPR41 in the phytantriol lipid system. The structural changes to the lipid matrix upon protein incorporation were greater for cholesterol-doped systems, potentially indicative of increased receptor uptake., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Amphiphilic compounds are used in a variety of applications due to their lyotropic liquid-crystalline phase formation, however only a limited number of compounds, in a potentially limitless field, are currently in use. A library of organic amphiphilic compounds was synthesised consisting of glucose, galactose, lactose, xylose and mannose head groups and double and triple-chain hydrophobic tails. A modular, high-throughput approach was developed, whereby head and tail components were conjugated using the copper-catalysed azide-alkyne cycloaddition (CuAAC) reaction. The tails were synthesised from two core alkyne-tethered intermediates, which were subsequently functionalised with hydrocarbon chains varying in length and degree of unsaturation and branching, while the five sugar head groups were selected with ranging substitution patterns and anomeric linkages. A library of 80 amphiphiles was subsequently produced, using a 24-vial array, with the majority formed in very good to excellent yields. A preliminary assessment of the liquid-crystalline phase behaviour is also presented.

A new method for the discovery of amphiphiles by using high-throughput (HT) methods to synthesise and characterise a library of galactose- and glucose-containing amphiphilic compounds is presented. The copper-catalysed azide–alkyne cycloaddition (CuAAC) “click” reaction between azide-tethered simple sugars and alkyne-substituted hydrophobic tails was employed to synthesise a library of compounds with systematic variations in chain length and unsaturation in a 24-vial array format. The liquid–crystalline phase behaviour was characterised in a HT manner by using synchrotron small-angle X-ray scattering (SSAXS). The observed structural variation with respect to chain parameters, including chain length and degree of unsaturation, is discussed, as well as hydration effects and degree of hydrogen bonding between head groups. The validity of our HT screening approach was verified by resynthesising a short-chain glucose amphiphile. A separate phase analysis of this compound confirmed the presence of numerous lyotropic liquid–crystalline phases.

Synthetic branched-chain glycolipids are suitable as model systems in understanding biological cell membranes, particularly because certain natural lipids possess chain branching. Herein, four branched-chain glycopyranosides, namely, 2-hexyl-decyl-α-D-glucopyranoside (α-Glc-OC10C6), 2-hexyl-decyl-β-D-glucopyranoside (β-Glc-OC10C6), 2-hexyl-decyl-α-D-galactopyranoside (α-Gal-OC10C6), and 2-hexyl-decyl-β-D-galactopyranoside (β-Gal-OC10C6), with a total alkyl chain length of 16 carbon atoms have been synthesized, and their phase behavior has been studied. The partial binary phase diagrams of these nonionic surfactants in water were investigated by optical polarizing microscopy (OPM) and small-angle X-ray scattering (SAXS). The introduction of chain branching in the hydrocarbon chain region is shown to result in the formation of inverse structures such as inverse hexagonal and inverse bicontinuous cubic phases. A comparison of the four compounds showed that they exhibited different polymorphism, especially in the thermotropic state, as a result of contributions from anomeric and epimeric effects according to their stereochemistry. The neat α-Glc-OC10C6 compound exhibited a lamellar (Lα) phase whereas dry α-Gal-OC10C6 formed an inverse bicontinuous cubic Ia3d (QII(G)) phase. Both β-anomers of glucoside and galactoside adopted the inverse hexagonal phase (HII) in the dry state. Generally, in the presence of water, all four glycolipids formed inverse bicontinuous cubic Ia3d (QII(G)) and Pn3m (QII(D)) phases over wide temperature and concentration ranges. The formation of inverse nonlamellar phases by these Guerbet branched-chain glycosides confirms their potential as materials for novel biotechnological applications such as drug delivery and crystallization of membrane proteins.

Amphiphile self-assembly materials, which contain both a hydrophilic and a hydrophobic domain, have great potential in high-throughput and combinatorial approaches to discovery and development. However, the materials chemistry community has not embraced these ideas to anywhere near the extent that the medicinal chemistry community has. While this situation is beginning to change, extracting the full potential of high-throughput approaches in the development of self-assembling materials will require further development in the synthesis, characterization, formulation, and application domains. One of the key factors that make small molecule amphiphiles prospective building blocks for next generation multifunctional materials is their ability to self-assemble into complex nanostructures through low-energy transformations. Scientists can potentially tune, control, and functionalize these structures, but only after establishing their inherent properties. Because both robotic materials handling and customized rapid characterization equipment are increasingly available, high-throughput solutions are now attainable. These address traditional development bottlenecks associated with self-assembling amphiphile materials, such as their structural characterization and the assessment of end-use functional performance. A high-throughput methodology can help streamline materials development workflows, in accord with existing high-throughput discovery pipelines such as those used by the pharmaceutical industry in drug discovery. Chemists have identified several areas that are amenable to a high-throughput approach for amphiphile self-assembly materials development. These allow an exploration of not only a large potential chemical, compositional, and structural space, but also material properties, formulation, and application variables. These areas of development include materials synthesis and preparation, formulation, characterization, and screening performance for the desired end application. High-throughput data analysis is crucial at all stages to keep pace with data collection. In this Account, we describe high-throughput advances in the field of amphiphile self-assembly, focusing on nanostructured lyotropic liquid crystalline materials, which form when amphiphiles are added to a polar solvent. We outline recent progress in the automated preparation of amphiphile molecules and their nanostructured self-assembly systems both in the bulk phase and in dispersed colloidal particulate systems. Once prepared, we can structurally characterize these systems by establishing phase behavior in a high-throughput manner with both laboratory (infrared and light polarization microscopy) and synchrotron facilities (small-angle X-ray scattering). Additionally, we provide three case studies to demonstrate how chemists can use high-throughput approaches to evaluate the functional performance of amphiphile self-assembly materials. The high-throughput methodology for the set-up and characterization of large matrix in meso membrane protein crystallization trials can illustrate an application of bulk phase self-assembling amphiphiles. For dispersed colloidal systems, two nanomedicine examples highlight advances in high-throughput preparation, characterization, and evaluation: drug delivery and magnetic resonance imaging agents.

The solid state and lyotropic phase behavior of a series of nonionic diethanolamide amphiphiles with increasing saturated hydrocarbon chain length (lauroyl, myristoyl, palmitoyl, and stearoyl) has been examined. All four saturated diethanolamide amphiphiles form a crystalline solid with two or three different polymorphic crystalline forms at room temperature. Melting points and associated enthalpies for these four amphiphiles increased with increasing chain length. Approximate partial binary phase diagrams have been constructed for each amphiphile/water system by combining Cross-Polarized Optical Microscopy (POM) and Small-Angle X-ray Scattering (SAXS) results. In the presence of water, all four diethanolamides form an L(α) phase, between 10% and 50% water content, and an L(2) phase with decreasing hydration and increasing temperature. In addition to the L(α) and L(2) phases, the shorter chain diethanolamide amphiphiles (lauroyl and myristoyl) also display a normal micellar phase (L(1)) at higher water contents, occurring to lower temperatures than the L(α) phase. By examining the effect of subtle molecular changes on both neat and lyotropic phase behavior, amphiphiles can be designed with properties tailored to a desired application., (Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.)

A protocol is presented for the high-throughput (HT) production of lyotropic liquid crystalline phases from libraries of lipids and lipid mixtures using standard liquid dispensing robotics, implementing methods that circumvent the problems traditionally associated with handling the highly viscous cubic phase. In addition, the ability to structurally characterize lipidic phases and assess functionality for membrane proteins contained within cubic phases, in a HT manner, is demonstrated. The techniques are combined and exemplified using the application of membrane protein crystallization within lipidic cubic phases.

The thermotropic and lyotropic liquid crystalline phase behaviour of a series of diethanolamide amphiphiles with isoprenoid-type hydrocarbon chains (geranoyl, H-farnesoyl, and phytanoyl) has been investigated. When neat, both H-farnesoyl and phytanoyl diethanolamide form a smectic liquid crystalline structure at sub-zero temperatures. In addition, all three diethanolamides exhibit a glass transition temperature at around -73 °C. Geranoyl diethanolamide forms a lamellar crystalline phase with a lattice parameter of 17.4 Å following long term storage accompanied by the loss of the glass transition. In the presence of water, H-farnesoyl and phytanoyl diethanolamide form lyotropic liquid crystalline phases, whilst geranoyl diethanolamide forms an L(2) phase. H-farnesoyl diethanolamide forms a fluid lamellar phase (L(α)) at room temperature and up to ∼ 40 °C. Phytanoyl diethanolamide displays a rich mesomorphism forming the inverse diamond (Q(II)(D)) and gyroid (Q(II)(G)) bicontinuous cubic phases in addition to an L(α) phase., (This journal is © the Owner Societies 2011)

The neat and lyotropic liquid crystalline phase behavior of three nonionic diethanolamide amphiphiles with C18 hydrocarbon chains containing one, two or three unsaturated bonds has been examined. This has allowed the effect of degree of unsaturation on the phase behavior of diethanolamide amphiphiles to be investigated. Neat linoleoyl and linolenoyl diethanolamide undergo a transition from a glassy liquid crystal to a liquid crystal at ∼-85 °C, while neat oleoyl diethanolamide undergoes a transition at ∼-60 °C to a liquid crystalline material before re-crystallizing at -34 °C. Oleoyl diethanolamide then undergoes a third transition from a crystalline phase to a smectic liquid crystalline phase at ∼5 °C. In the absence of water, the transition temperature from a smectic liquid crystal to an isotropic liquid decreases with increasing unsaturation. The addition of water results in the formation of a lamellar phase (L(α)) for all three amphiphiles. The lamellar phase is stable under excess water conditions up to temperatures of at least 70 °C. Approximate partial binary amphiphile-water phase diagrams generated for the three unsaturated C18 amphiphiles indicate that the excess water point for each amphiphile occurs at ∼60% (w/w) amphiphile., (This journal is © the Owner Societies 2011)

An amphiphile prodrug, 5'-deoxy-5-fluoro-N(4)-(palmityloxycarbonyl) cytidine or 5'-deoxy-5-fluoro-N(4)-(hexadecanaloxycarbonyl) cytidine (5-FCPal), consisting of the same head group as the commercially available chemotherapeutic agent Capecitabine, linked to a palmityl hydrocarbon chain via a carbamate bond is reported. Thermal analysis of this prodrug indicates that it melts at ∼115 °C followed quickly by degradation beginning at ∼120 °C. The neat solid 5-FCPal amphiphile acquires a lamellar crystalline arrangement with a d-spacing of 28.6±0.3 Å, indicating interdigitation of the hydrocarbon chains. Under aqueous conditions, solid 5-FCPal is non-swelling and no lyotropic liquid crystalline phase formation is observed. In order to assess the in vitro toxicity and in vivo efficacy in colloidal form, solid lipid nanoparticles (SLNs) with an average size of ∼700 nm were produced via high pressure homogenization. The in vitro toxicity of the 5-FCPal SLNs against several different cancer and normal cell types was assessed over a 48 h period, and IC(50) values were comparable to those observed for Capecitabine. The in vivo efficacy of the 5-FCPal SLNs was then assessed against the highly aggressive mouse 4T1 breast cancer model. To do so, the prodrug SLNs were administered orally at 3 different dosages (0.1, 0.25, 0.5 mmol/mouse/day) and compared to Capecitabine delivered at the same dosages. After 21 days of receiving the treatments, the 0.5 mmol dose of 5-FCPal exhibited the smallest average tumour volume. Since 5-FCPal is activated in a similar manner to Capecitabine via a 3 step enzymatic pathway with the final step occurring preferentially at the tumour site, formulation of the prodrug into SLNs combines the advantage of selective, localized activation with the sustained release properties of nanostructured amphiphile self-assembly and multiple payload materials thereby potentially creating a more effective anticancer agent., (Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.)

An amphiphile prodrug, 5'-deoxy-5-fluoro-N⁴-(phytanyloxycarbonyl) cytidine (5-FCPhy) has been prepared and investigated for its self-assembly material properties, in vitro cytotoxicity, and in vivo efficacy as a chemotherapy agent. The phase transitions and stability of the neat amphiphile were characterized by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). X-ray diffraction (XRD) was used to confirm the structure of the neat amphiphile, which was an amorphous glassy material. The lyotropic liquid crystalline self-assembly behavior of the amphiphile prodrug in water was examined by cross polarizing optical microscopy (POM) and small-angle X-ray scattering (SAXS). Under excess water conditions at room temperature, the amphiphile prodrug self-assembles into lyotropic liquid crystalline mesophases of inverse bicontinuous cubic symmetry. Upon aging, the inverse cubic phase slowly transformed to an inverse hexagonal phase. This amphiphile was successfully dispersed into nanoparticles of cubic and hexagonal symmetry. The in vitro cytotoxicity of dispersed nanoparticles was evaluated in seven different normal and cancer cell types and exhibited IC₅₀ values between 70 and 90 μM for all cell types. Evaluation of 5-FCPhy in vivo against a mouse 4T1 breast tumor model displayed a trend of increasing efficacy with increasing dose. Furthermore, after 21 days, tumor volumes in the 0.5 mmol 5-FCPhy treatment group were significantly smaller than all other treatment groups including mice receiving a short chain water-soluble analogue, Capecitabine (a commercially available oral chemotherapy agent), delivered at the same dosage.

We demonstrate that oral delivery of self-assembled nanostructured nanoparticles consisting of 5-fluorouracil (5-FU) lipid prodrugs results in a highly effective, target-activated, chemotherapeutic agent, and offers significantly enhanced efficacy over a commercially available alternative that does not self-assemble. The lipid prodrug nanoparticles have been found to significantly slow the growth of a highly aggressive mouse 4T1 breast tumour, and essentially halt the growth of a human MDA-MB-231 breast tumour in mouse xenografts. Systemic toxicity is avoided as prodrug activation requires a three-step, enzymatic conversion to 5-FU, with the third step occurring preferentially at the tumour site. Additionally, differences in the lipid prodrug chemical structure and internal nanostructure of the nanoparticle dictate the enzymatic conversion rate and can be used to control sustained release profiles. Thus, we have developed novel oral nanomedicines that combine sustained release properties with target-selective activation.

Three nucleoside lipids have been synthesized: 3'-oleoylthymidine, 3',5'-dioleoylthymidine, and 3'-phytanoylthymidine. Differential scanning calorimetry and X-ray diffraction have been employed to characterize the physical properties of these neat lipids. Polarizing optical microscopy, small-angle X-ray scattering, and cryo-transmission electron microscopy techniques have been used to investigate the phase behavior in aqueous systems. Both oleoyl-based nucleoside lipids adopted a lamellar crystalline phase in the neat form at room temperature, and the phytanoyl derivative exhibited a fluid isotropic phase. Under excess water conditions, the presence of one branched (phytanoyl) or one unsaturated (oleoyl) chain promoted the formation of a liquid-crystalline lamellar phase at physiological temperatures. In contrast, the 3',5'-dioleoylthymidine derivative is nonswelling and does not exhibit lyotropic liquid-crystalline phase behavior. The nucleolipids' propensity for DNA-type binding and recognition has been evaluated by using a monolayer system to measure surface pressure-area isotherms in a Langmuir trough and indicates that the nucleoside base is available for nonspecific hydrogen bonding in the monolayer liquid expanded state for the single-chain nucleolipids but not for the dual-chain amphiphile.

The preparation, characterisation and assessment of drug delivery vehicles is typically a slow and complex process. Here we present a nanostructured nanoparticle system that can be prepared and characterised in a high-throughput fashion. In particular we use phytantriol and Myverol to prepare inverse bicontinuous cubic and inverse hexagonal liquid crystalline nanoparticles loaded with 10 commonly used therapeutic agents at increasing concentration. The dispersions are prepared using automated apparatus to create different concentrations and phases using novel protocols. We are able to characterise each stabilised nanoparticle dispersion using a range of methodologies including small angle X-ray scattering, particle sizing and drug partitioning. With this information we are able to assess which drug delivery vehicle is preferred for each drug and at which concentration the drug should be loaded to ensure maximum payload and to retain particle integrity., (Crown Copyright (c) 2010. Published by Elsevier B.V. All rights reserved.)

Lanthanide salts of phytanic acid, an isoprenoid-type amphiphile, have been synthesized and characterized. Elemental analysis and FTIR spectroscopy were used to confirm the formed product and showed that three phytanate anions are complexed with one lanthanide cation. The physicochemical properties of the lanthanide phytanates were investigated using DSC, XRD, SAXS, and cross-polarized optical microscopy. Several of the hydrated salts form a liquid-crystalline hexagonal columnar mesophase at room temperature, and samarium(III) phytanate forms this phase even in the absence of water. Select lanthanide phytanates were dispersed in water, and cryo-TEM images indicate that some structure has been retained in the dispersed phase. NMR relaxivity measurements were conducted on these systems. It has been shown that a particulate dispersion of gadolinium(III) phytanate displays proton relaxivity values comparable to those of a commercial contrast agent for magnetic resonance imaging and a colloidal dispersion of europium(III) phytanate exhibits the characteristics of a fluorescence imaging agent.

The self-assembly, solid state and lyotropic liquid crystalline phase behavior of a series of endogenous n-acylethanolamides (NAEs) with differing degrees of unsaturation, viz., oleoyl monoethanolamide, linoleoyl monoethanolamide, and linolenoyl monoethanolamide, have been examined. The studied molecules are known to possess inherent biological function. Both the monoethanolamide headgroup and the unsaturated hydrophobe are found to be important in dictating the self-assembly behavior of these molecules. In addition, all three molecules form lyotropic liquid crystalline phases in water, including the inverse bicontinuous cubic diamond (Q(II)(D)) and gyroid (Q(II)(G)) phases. The ability of the NAE's to form inverse cubic phases and to be dispersed into ordered nanostructured colloidal particles, cubosomes, in excess water, combined with their endogenous nature and natural medicinal properties, makes this new class of soft mesoporous amphiphile self-assembly materials suitable candidates for investigation in a variety of advanced multifunctional applications, including encapsulation and controlled release of therapeutic agents and incorporation of medical imaging agents.

Gadolinium oleate has been added at various concentrations to a Myverol inverse bicontinuous cubic phase forming system, and the potential of these systems as magnetic resonance imaging (MRI) contrast agents has been investigated. Differential scanning calorimetry (DSC), small-angle X-ray scattering (SAXS), and cryo-transmission electron microscopy (cryo-TEM) measurements on the Gd oleate/Myverol systems indicate that Gd oleate is at least partially incorporated within the cubic phase of Myverol. However, at Gd oleate concentrations greater than 1 wt %, partial phase separation of the system may occur with the formation of a Gd-oleate-rich lamellar phase as well as the cubic phase. Bulk Gd oleate/Myverol mixtures can be dispersed into stable colloidal dispersions. SAXS and cryo-TEM measurements on these dispersions indicate that the presence of Gd oleate in the Myverol system prevents the formation of cubosomes from the bulk cubic phase. Instead, the dispersion consists of putative Gd-oleate-rich nonswelling lamellar nanoparticles as well as colloidal particles lacking ordered internal structure. In vitro studies on these dispersions demonstrated that the relaxivity of select Gd oleate/Myverol systems is much higher than that of pure Gd oleate, exemplifying the promise of this system type for magnetic resonance imaging. The highest water proton relaxivities (r(1) = 34.2 mM(-1) s(-1) and r(2) = 27.3 mM(-1) s(-1) at 20 MHz and room temperature) were obtained at a Gd oleate loading concentration of 1 wt %, with a subsequent decrease in relaxivity with increasing Gd oleate concentration. These maximum relaxivities compare favorably with the relaxivities for the commercial contrast agent, Magnevist (r(1) = 4.91 mM(-1) s(-1) and r(2) = 6.26 mM(-1) s(-1) at 20 MHz and room temperature).

The n-acylethanolamides (NAEs) are a family of naturally occurring monoethanolamide containing lipids that display a variety of interesting biological properties. In this study, some physicochemical properties of a series of saturated monoethanolamide lipids with increasing hydrocarbon chain length (lauroyl, myristoyl, palmitoyl, and stearoyl) have been investigated. Temperature induced phase transitions for these NAEs indicate that both the monoethanolamide headgroups and the unsaturated hydrophobic tails play a role in the melting behavior of these lipids. All four lipids examined demonstrate the presence of at least three different polymorphic crystal forms. Transitions in crystal structure can be induced via heating and visualized with polarized optical microscopy. At room and physiological temperature, the four NAEs are solid lamellar crystalline materials. All four molecules form lyotropic liquid crystalline phases in water, albeit at relatively high temperatures, including the lamellar liquid crystalline phase and at least two isotropic phases. Lamellar crystalline palmitoyl monoethanolamide was dispersed as solid lipid nanoparticles (SLNs). The cytotoxicity of these SLNs toward human mammary epithelial cells (HMEpiC) and the MCF7 breast cancer cell line was assessed at physiological temperature. The palmitoyl monoethanolamide SLNs showed little to no toxicity to the HMEpiC even at a concentration of 30 microM. At concentrations above 3 microM, the HMEpiC population was reduced by less than 15%, while the MCF7 population was reduced by approximately 20-30%. The endogenous nature and natural medicinal properties make this series of lipids ideal candidates for further investigation as solid lipid nanoparticle drug delivery systems.

Eight lanthanide(III) oleates have been prepared and characterized. The chelation and self-assembly structures of these rare-earth oleates have been studied by elemental analysis, Fourier transfer infrared spectroscopy (FTIR), and X-ray powder diffraction (XRD) analysis. Elemental analysis and FTIR results indicate that three oleate anions are complexed with one lanthanide cation and, with the exception of anhydrous cerium(III) oleate, form either a mono- or a hemihydrate. The X-ray analysis showed that the neat lanthanide soaps have a lamellar bilayer structure at room temperature. The thermal behavior has been investigated by cross-polarized optical microscopy (POM), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). POM scans showed that all the lanthanide oleates form a lamellar phase in the presence of excess water. Small-angle X-ray scattering (SAXS) and XRD were used to investigate the internal structure of the bulk lanthanide oleates in excess water, and these X-ray results confirmed that the lanthanide oleates do not swell in water. Select lanthanide oleates were dispersed in water to form nonswelling lamellar submicrometer particles, confirmed by dynamic light scattering (DLS) and synchrotron SAXS measurements. NMR results indicated that colloidal dispersions of lanthanide oleates containing paramagnetic ions, such as gadolinium(III), terbium(III), and dysprosium(III), have a significant effect on the longitudinal (T(1)) and transverse (T(2)) relaxation times of protons in water. Time-resolved fluorescence measurements have demonstrated that colloidal dispersions of europium(III) oleate exhibit strong luminescence. The rare earth metal soaps exemplify the potential of self-assembled chelating amphiphiles as contrast agents in medical imaging modalities such as magnetic resonance imaging (MRI) and fluorescence imaging.

In the past two decades, the geometric pathways involved in the transformations between inverse bicontinuous cubic phases in amphiphilic systems have been extensively theoretically modeled. However, little experimental data exists on the cubic-cubic transformation in pure lipid systems. We have used pressure-jump time-resolved X-ray diffraction to investigate the transition between the gyroid QGII and double-diamond QDII phases in mixtures of 1-monoolein in 30 wt % water. We find for this system that the cubic-cubic transition occurs without any detectable intermediate structures. In addition, we have determined the kinetics of the transition, in both the forward and reverse directions, as a function of pressure-jump amplitude, temperature, and water content. A recently developed model allows (at least in principle) the calculation of the activation energy for lipid phase transitions from such data. The analysis is applicable only if kinetic reproducibility is achieved, at least within one sample, and achievement of such kinetic reproducibility is shown here, by carrying out prolonged pressure-cycling. The rate of transformation shows clear and consistent trends with pressure-jump amplitude, temperature, and water content, all of which are shown to be in agreement with the effect of the shift in the position of the cubic-cubic phase boundary following a change in the thermodynamic parameters.

Inverse bicontinuous cubic lyotropic phases are a complex solution to the dilemma faced by all self-assembled water-amphiphile systems: how to satisfy the incompatible requirements for uniform interfacial curvature and uniform molecular packing. The solution reached in this case is for the water-amphiphile interfaces to deform hyperbolically onto triply periodic minimal surfaces. We have previously suggested that although the molecular packing in these structures is rather uniform the relative phase behavior of the gyroid, double diamond, and primitive inverse bicontinuous cubic phases can be understood in terms of subtle differences in packing frustration. In this work, we have calculated the packing frustration for these cubics under the constraint that their interfaces have constant mean curvature. We find that the relative packing stress does indeed differ between phases. The gyroid cubic has the least packing stress, and at low water volume fraction, the primitive cubic has the greatest packing stress. However, at very high water volume fraction, the double diamond cubic becomes the structure with the greatest packing stress. We have tested the model in two ways. For a system with a double diamond cubic phase in excess water, the addition of a hydrophobe may release packing frustration and preferentially stabilize the primitive cubic, since this has previously been shown to have lower curvature elastic energy. We have confirmed this prediction by adding the long chain alkane tricosane to 1-monoolein in excess water. The model also predicts that if one were able to hydrate the double diamond cubic to high water volume fractions, one should destabilize the phase with respect to the primitive cubic. We have found that such highly swollen metastable bicontinuous cubic phases can be formed within onion vesicles. Data from monoelaidin in excess water display a well-defined transition, with the primitive cubic appearing above a water volume fraction of 0.75. Both of these results lend support to the proposition that differences in the packing frustration between inverse bicontinuous cubic phases play a pivotal role in their relative phase stability.

In this paper, we give an overview of our studies by static and time-resolved X-ray diffraction of inverse cubic phases and phase transitions in lipids. In [section sign] 1, we briefly discuss the lyotropic phase behaviour of lipids, focusing attention on non-lamellar structures, and their geometric/topological relationship to fusion processes in lipid membranes. Possible pathways for transitions between different cubic phases are also outlined. In [section sign] 2, we discuss the effects of hydrostatic pressure on lipid membranes and lipid phase transitions, and describe how the parameters required to predict the pressure dependence of lipid phase transition temperatures can be conveniently measured. We review some earlier results of inverse bicontinuous cubic phases from our laboratory, showing effects such as pressure-induced formation and swelling. In [section sign] 3, we describe the technique of pressure-jump synchrotron X-ray diffraction. We present results that have been obtained from the lipid system 1:2 dilauroylphosphatidylcholine/lauric acid for cubic-inverse hexagonal, cubic-cubic and lamellar-cubic transitions. The rate of transition was found to increase with the amplitude of the pressure-jump and with increasing temperature. Evidence for intermediate structures occurring transiently during the transitions was also obtained. In [section sign] 4, we describe an IDL-based 'AXcess' software package being developed in our laboratory to permit batch processing and analysis of the large X-ray datasets produced by pressure-jump synchrotron experiments. In [section sign] 5, we present some recent results on the fluid lamellar-Pn3m cubic phase transition of the single-chain lipid 1-monoelaidin, which we have studied both by pressure-jump and temperature-jump X-ray diffraction. Finally, in [section sign] 6, we give a few indicators of future directions of this research. We anticipate that the most useful technical advance will be the development of pressure-jump apparatus on the microsecond time-scale, which will involve the use of a stack of piezoelectric pressure actuators. The pressure-jump technique is not restricted to lipid phase transitions, but can be used to study a wide range of soft matter transitions, ranging from protein unfolding and DNA unwinding and transitions, to phase transitions in thermotropic liquid crystals, surfactants and block copolymers.

The liquid crystalline lamellar (L(alpha)) to double-diamond inverse bicontinuous cubic (Q(D)(pi)) phase transition for the amphiphile monoelaidin in excess water exhibits a remarkable sequence of structural transformations for pressure or temperature jumps. Our data imply that the transition dynamics depends on a coupling between changes in molecular shape and the geometrical and topological constraints of domain size. We propose a qualitative model for this coupling based on theories of membrane fusion via stalks and existing knowledge of the structure and energetics of bicontinuous cubic phases.