A deep eutectic solvent is an effective cryoprotective agent for platelets.

The most widely used method of platelet cryopreservation requires the addition of dimethyl sulfoxide (DMSO; Me2SO) as a cryoprotective agent (CPA) and pre-freeze removal of Me2SO before freezing to mitigate toxicity. However, alternative CPAs such as deep eutectic solvents (DES), which are less toxic could simplify this process. The aim of this study was to determine the effectiveness of a Proline-Glycerol (Prol-Gly 1:3) DES as a platelet CPA. Platelets were cryopreserved at -80 °C using 10 % Prol-Gly 1:3 (DES; n = 6), or in the absence of a cryoprotectant (no CPA; n = 6). Platelets were also cryopreserved according to the gold-standard blood-banking method using 5.5 % Me2SO (n = 6), with centrifugation and pre-freeze removal of the excess Me2SO. Platelet quality was assessed by flow cytometry and thromboelastography (TEG). Post-thaw recovery was similar between the three groups. The abundance of labile platelet glycoproteins GPIbα and GPVI were highest in the DES group, however, markers of activation (CD62P and annexin-V) were also higher in this group. In terms of function, the strength of the clot (maximum amplitude; TEG) and extent of clot retraction was better with DES platelets compared to no CPA, but lower than Me2SO platelets. DES provides a cryoprotective advantage to platelets when compared to no CPA. Importantly, when compared to Me2SO platelets, most quality parameters were similar in DES platelets. The major advantage with using a DES is biocompatibility, therefore it does not need to be removed prior to transfusion. This greatly simplifies the freezing and thawing process, avoiding the toxic effects of Me2SO.

Selective ion transport across a lipid bilayer in a protic ionic liquid.

Ionic liquids (ILs) have exhibited enormous potential as electrolytes, designer solvents and reaction media, as well as being surprisingly effective platforms for amphiphile self-assembly and for preserving structure of complex biomolecules. This has led to their exploration as media for long-term biopreservation and in biosensors, for which their viability depends on their ability to sustain both structure and function within complex, multicomponent nanoscale compartments and assemblies. Here we show that a tethered lipid bilayer can be assembled directly in a purely IL environment that retains its structure upon exchange between IL and aqueous buffer, and that the membrane transporter valinomycin can be incorporated so as to retain its functionality and cation selectivity. This paves the way for the development of long-lived, non-aqueous microreactors and sensor assemblies, and demonstrates the potential for complex proteins to retain functionality in non-aqueous, ionic liquid solvents.

Analysis of Pathogenic Bacterial and Yeast Biofilms Using the Combination of Synchrotron ATR-FTIR Microspectroscopy and Chemometric Approaches.

Biofilms are assemblages of microbial cells, extracellular polymeric substances (EPS), and other components extracted from the environment in which they develop. Within biofilms, the spatial distribution of these components can vary. Here we present a fundamental characterization study to show differences between biofilms formed by Gram-positive methicillin-resistant Staphylococcus aureus (MRSA), Gram-negative Pseudomonas aeruginosa, and the yeast-type Candida albicans using synchrotron macro attenuated total reflectance-Fourier transform infrared (ATR-FTIR) microspectroscopy. We were able to characterise the pathogenic biofilms' heterogeneous distribution, which is challenging to do using traditional techniques. Multivariate analyses revealed that the polysaccharides area (1200-950 cm-1) accounted for the most significant variance between biofilm samples, and other spectral regions corresponding to amides, lipids, and polysaccharides all contributed to sample variation. In general, this study will advance our understanding of microbial biofilms and serve as a model for future research on how to use synchrotron source ATR-FTIR microspectroscopy to analyse their variations and spatial arrangements.

Filler size effect in an attractive fibrillated network: a structural and rheological perspective.

The effect of the filler size on the structural and mechanical properties of an attractive fibrillated network composed of oxidised cellulose nanofibrils (OCNF) in water was investigated. Silica nanoparticles with a diameter of ca. 5 nm (SiNp5) and and ca. 158 nm (SiNp158) were chosen as non-interacting fillers of the OCNF network. These filler sizes were chosen, respectively, to have a particle size which was either similar to that of the network mesh size or much larger than it. Contrast matched small angle neutron scattering (SANS) experiments revealed that the presence of the fillers (SiNp5 and SiNp158) did not perturb the structural properties of the OCNF network at the nanometer scale. However, the filler size difference strongly affected the mechanical properties of the hydrogel upon large amplitude oscillatory shear. The presence of the smaller filler, SiNp5, preserved the mechanical properties of the hydrogels, while the larger filler, SiNp158, allowed a smoother breakage of the network and low network recoverability after breakage. This study showed that the filler-to-mesh size ratio, for non-interacting fillers, is pivotal for tailoring the non-linear mechanical properties of the gel, such as yielding and flow.

Impact of wormlike micelles on nano and macroscopic structure of TEMPO-oxidized cellulose nanofibril hydrogels.

In this work, we investigated the effect of adding surfactant mixtures on the rheological properties of TEMPO-oxidized cellulose nanofibril (OCNF) saline dispersions. Three surfactant mixtures were studied: cocamidopropyl betaine (CAPB)/sodium dodecyl sulfate (SDS), which forms wormlike micelles (WLMs); cocamidopropylamine oxide (CAPOx)/SDS, which forms long rods; and CAPB/sodium lauroyl sarcosinate (SLS), which forms spherical micelles. The presence of micelles in these surfactant mixtures, independent of their morphology, leads to an increase of tan δ, making the gels less solid-like, therefore acting as a plasticizer. WLMs were able to suppress strain stiffening normally observed in OCNF gels at large strains. OCNF/WLM gels have lower G' values than OCNF gels while the other micellar morphologies have a reduced impact on G'. The presence of unconnected micelles leads to increased dissipative deformation in OCNF gels without affecting the connectivity of the fibrils, while the presence of entangled micelles interferes with the OCNF network.

Effect of Deep Eutectic Solvent Nanostructure on Phospholipid Bilayer Phases.

Phospholipids are shown by solvent penetration experiments to form lamellar phases and spontaneously spawn vesicles in a wide range of deep eutectic solvents (DESs) composed of alkylammonium halide salts and glycerol or ethylene glycol, which are shown to be nanostructured by X-ray scattering. In contrast with molecular solvents, the chain melting temperature of each phospholipid, which determines the stability of the swellable bilayer phase, depends on the structure of the cation, anion, and molecular H-bond donor that constitute the DES. Chain melting is most sensitive to the length of the alkyl chain of the cation, which is partitioned between apolar domains in the bulk, nanostructured DES and those within the lipid bilayer. This is moderated by the structures of the anion and the molecular hydrogen bond donor, which determine the extent of polar/apolar segregation in the bulk liquid.

Effect of protic ionic liquid nanostructure on phospholipid vesicle formation.

The formation of bilayer-based lyotropic liquid crystals and vesicle dispersions by phospholipids in a range of protic ionic liquids has been investigated by polarizing optical microscopy using isothermal penetration scans, differential scanning calorimetry, and small angle X-ray and neutron scattering. The stability and structure of both lamellar phases and vesicle dispersions is found to depend primarily on the underlying amphiphilic nanostructure of the ionic liquid itself. This finding has significant implications for the use of ionic liquids in soft and biological materials and for biopreservation, and demonstrates how vesicle structure and properties can be controlled through selection of cation and anion. For a given ionic liquid, systematic trends in bilayer thickness, chain-melting temperature and enthalpy increase with phospholipid acyl chain length, paralleling behaviour in aqueous systems.

Spontaneous vesicle formation in a deep eutectic solvent.

Solvent penetration experiments and small-angle X-ray scattering reveal that phospholipids dissolved in a deep eutectic solvent (DES) spontaneously self-assemble into vesicles above the lipid chain melting temperature. This means DESs are one of the few nonaqueous solvents that mediate amphiphile self-assembly, joining a select set of H-bonding molecular solvents and ionic liquids.

Molecular interactions with bilayer membrane stacks using neutron and X-ray diffraction.

Lamellar unit cell reconstruction from neutron and X-ray diffraction data provides information about the disposition and position of molecules and molecular segments with respect to the bilayer. When supplemented with the judicious use of molecular deuteration, the technique probes the molecular interactions and conformations within the bilayer membrane and the water layer which constitute the crystallographic unit cell. The perspective is model independent, and potentially, with a higher degree of resolution than is available with other techniques. In the case of neutron diffraction the measurement consists of carefully normalised diffracted intensity under conditions of contrast variation of the water layer. The subsequent Fourier reconstruction of the unit cell is made using the phase information from variation of peak intensities with contrast. Although the phase problem is not as easily solved for the corresponding X-ray measurements, an intuitive approach can often suffice. Here we discuss the two complimentary techniques as probes of scattering length density profiles of a bilayer, and how such a perspective provides information about the location and orientation of molecules within or between lipid bilayers. Within the basic paradigm of lamellar phases this method has provided, for example, detailed insights into the location and interaction of cryoprotectants and stress proteins, of the mechanisms of actions of viral proteins, antimicrobial compounds and drugs, and the underlying structure of the stratum corneum. In this paper we review these techniques and provide examples of the systems that have been examined. We finish with a future outlook on the use of these techniques to improve our understanding of the interactions of membranes with biomolecules.

Deep Eutectic Solvent Eutectogels for Delivery of Broad-Spectrum Antimicrobials.

Gel-based wound dressings have gained popularity within the healthcare industry for the prevention and treatment of bacterial and fungal infections. Gels based on deep eutectic solvents (DESs), known as eutectogels, provide a promising alternative to hydrogels as they are non-volatile and highly tunable and can solubilize therapeutic agents, including those insoluble in hydrogels. A choline chloride:glycerol-cellulose eutectogel was loaded with numerous antimicrobial agents including silver nanoparticles, black phosphorus nanoflakes, and commercially available pharmaceuticals (octenidine dihydrochloride, tetracycline hydrochloride, and fluconazole). The eutectogels caused >97% growth reduction in Gram-positive methicillin-resistant Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa bacteria and the fungal species Candida albicans.

Scattering approaches to unravel protein solution behaviors in ionic liquids and deep eutectic solvents: From basic principles to recent developments.

Proteins in ionic liquids (ILs) and deep eutectic solvents (DESs) have gained significant attention due to their potential applications in various fields, including biocatalysis, bioseparation, biomolecular delivery, and structural biology. Scattering approaches including dynamic light scattering (DLS) and small-angle X-ray and neutron scattering (SAXS and SANS) have been used to understand the solution behavior of proteins at the nanoscale and microscale. This review provides a thorough exploration of the application of these scattering techniques to elucidate protein properties in ILs and DESs. Specifically, the review begins with the theoretical foundations of the relevant scattering approaches and describes the essential solvent properties of ILs and DESs linked to scattering such as refractive index, scattering length density, ion-pairs, liquid nanostructure, solvent aggregation, and specific ion effects. Next, a detailed introduction is provided on protein properties such as type, concentration, size, flexibility and structure as observed through scattering methodologies. This is followed by a review of the literature on the use of scattering for proteins in ILs and DESs. It is highlighted that enhanced data analysis and modeling tools are necessary for assessing protein flexibility and structure, and for understanding protein hydration, aggregation and specific ion effects. It is also noted that complementary approaches are recommended for comprehensively understanding the behavior of proteins in solution due to the complex interplay of factors, including ion-binding, dynamic hydration, intermolecular interactions, and specific ion effects. Finally, the challenges and potential research directions for this field are proposed, including experimental design, data analysis approaches, and supporting methods to obtain fundamental understandings of complex protein behavior and protein systems in solution. We envisage that this review will support further studies of protein interface science, and in particular studies on solvent and ion effects on proteins.

Biophysical Characterization and Cryopreservation of Mammalian Cells Using Ionic Liquids.

Ionic liquids (ILs) are a diverse class of solvents which can be selected for task-specific properties, making them attractive alternatives to traditional solvents. To tailor ILs for specific biological applications, it is necessary to understand the structure-property relationships of ILs and their interactions with cells. Here, a selection of carboxylate anion-based ILs were investigated as cryoprotectants, which are compounds added to cells before freezing to mitigate lethal freezing damage. The cytotoxicity, cell permeability, thermal behavior, and cryoprotective efficacy of the ILs were assessed with two model mammalian cell lines. We found that the biophysical interactions, including permeability of the ILs, were influenced by considering the IL pair together, rather than as single species acting independently. All of the ILs tested had high cytotoxicity, but ethylammonium acetate demonstrated good cryoprotective efficacy for both cell types tested. These results demonstrate that despite toxicity, ILs may be suitable for certain biological applications. It also demonstrates that more research is required to understand the contribution of ion pairs to structure-property relationships and that knowing the behavior of a single ionic species will not necessarily predict its behavior as part of an IL.

Phytantriol phase behaviour in choline chloride urea and water mixtures.

Deep eutectic solvents (DES) are tailorable non-aqueous solvents with promising properties for a range of applications, from industrial dissolution of plant products to biomedicine. They are mixtures of hydrogen bond donors and acceptors with low melting points that can be tailored to specific applications, and many support the self-assembly of amphiphilic molecules into lyotropic liquid crystal phases. Self-assembled lipid structures have potential for numerous applications, including drug delivery. These ordered structures can act as carriers, slow-release vehicles, or microreactors. Lipid self-assembly in non-aqueous solvents, such as deep eutectic solvents, is important for applications at extreme temperatures, or involving water-insoluble or water sensitive components. However, lipid self-assembly in these solvents remains largely unexplored. In this paper, we have examined the self-assembly of phytantriol, a non-ionic lipid, at 10 and 30 wt% in the deep eutectic solvent choline chloride:urea, with and without water. Self-assembly was assessed using small angle X-ray scattering and cross polarised optical microscopy at temperatures from 25-66 °C. We found that pure choline chloride:urea supports a Pn3m cubic phase similar to that formed in water. However, mixtures of the DES with water resulted in phytantriol forming an inverse hexagonal phase and influenced the phase transition temperatures. These results demonstrate that choline chloride:urea can support diverse phase behaviour, and also provides a mechanism for tailoring the phase for particular applications simply by controlling the amount of water in the solvent. In the future this could lead to methods of triggered release of drugs and biomolecules by the simple addition of water which could be critical for drug delivery applications.

Phase separation in a ternary DPPC/DOPC/POPC system with reducing hydration.

The maintenance of plasma membrane structure is vital for the viability of cells. Disruption of this structure can lead to cell death. One important example is the macroscopic phase separation observed during dehydration associated with desiccation and freezing, often leading to loss of permeability and cell death. It has previously been shown that the hybrid lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) can act as a line-active component in ternary lipid systems, inhibiting macroscopic phase separation and stabilising membrane microdomains in lipid vesicles [1]. The domain size is found to decrease with increasing POPC concentration until complete mixing is observed. However, no such studies have been carried out at reduced hydration. To examine if this phase separation is unique to vesicles in excess water, we have conducted studies on several binary and ternary model membrane systems at both reduced hydration ("powder" type samples and oriented membrane stacks) and in excess water (supported lipid bilayers) at 0.2 mol fraction POPC, in the range where microdomain stabilisation is reported. Differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FTIR) are used to map phase transition temperatures, with X-ray and neutron scattering providing details of the changes in lipid packing and phase information within these boundaries. Atomic force microscopy (AFM) is used to image bilayers on a substrate in excess water. In all cases, macroscopic phase separation was observed rather than microdomain formation at this molar ratio. Thus POPC does not stabilise microdomains under these conditions, regardless of the type of model membrane, hydration or temperature. Thus we conclude that the driving force for separation under these conditions overcomes any linactant effects of the hybrid lipid.

Bulk and interfacial nanostructure and properties in deep eutectic solvents: Current perspectives and future directions.

Deep eutectic solvents (DESs) are a tailorable class of solvents that are rapidly gaining scientific and industrial interest. This is because they are distinct from conventional molecular solvents, inherently tuneable via careful selection of constituents, and possess many attractive properties for applications, including catalysis, chemical extraction, reaction media, novel lubricants, materials chemistry, and electrochemistry. DESs are a class of solvents composed solely of hydrogen bond donors and acceptors with a melting point lower than the individual components and are often fluidic at room temperature. A unique feature of DESs is that they possess distinct bulk liquid and interfacial nanostructure, which results from intra- and inter-molecular interactions, including coulomb forces, hydrogen bonding, van der Waals interactions, electrostatics, dispersion forces, and apolar-polar segregation. This nanostructure manifests as preferential spatial arrangements of the different species, and exists over several length scales, from molecular- to nano- and meso-scales. The physicochemical properties of DESs are dictated by structure-property relationships; however, there is a significant gap in our understanding of the underlying factors which govern their solvent properties. This is a major limitation of DES-based technologies, as nanostructure can significantly influence physical properties and thus potential applications. This perspective provides an overview of the current state of knowledge of DES nanostructure, both in the bulk liquid and at solid interfaces. We provide definitions which clearly distinguish DESs as a unique solvent class, rather than a subset of ILs. An appraisal of recent work provides hints towards trends in structure-property relationships, while also highlighting inconsistencies within the literature suggesting new research directions for the field. It is hoped that this review will provide insight into DES nanostructure, their potential applications, and development of a robust framework for systematic investigation moving forward.

Deep eutectic solvents as cryoprotective agents for mammalian cells.

Cryopreservation has facilitated numerous breakthroughs including assisted reproductive technology, stem cell therapies, and species preservation. Successful cryopreservation requires the addition of cryoprotective agents to protect against freezing damage and dehydration. For decades, cryopreservation has largely relied on the same two primary agents: dimethylsulfoxide and glycerol. However, both of these are toxic which limits their use for cells destined for clinical applications. Furthermore, these two agents are ineffective for hundreds of cell types, and organ and tissue preservation has not been achieved. The research presented here shows that deep eutectic solvents can be used as cryoprotectants. Six deep eutectic solvents were explored for their cryoprotective capacity towards mammalian cells. The solvents were tested for their thermal properties, including glass transitions, toxicity, and permeability into mammalian cells. A deep eutectic solvent made from proline and glycerol was an effective cryoprotective agent for all four cell types tested, even with extended incubation prior to freezing. This deep eutectic solvent was more effective and less toxic than its individual components, highlighting the importance of multi-component systems. Cells were characterised post-thawing using atomic force microscopy and confocal microscopy. Molecular dynamics simulations support the biophysical parameters obtained by experimentation. This is one of the first times that this class of solvents has been systematically tested for cryopreservation of mammalian cells and as such this research opens the way for the development of potentially thousands of new cryoprotective agents that can be tailored to specific cell types. The demonstrated capacity of cells to be incubated with the deep eutectic solvent at 37 °C for hours prior to freezing without significant loss of viability is a major step toward the storage of organs and tissues.

Illuminating the biochemical interaction of antimicrobial few-layer black phosphorus with microbial cells using synchrotron macro-ATR-FTIR.

In the fight against drug-resistant pathogenic bacterial and fungal cells, low-dimensional materials are emerging as a promising alternative treatment method. Specifically, few-layer black phosphorus (BP) has demonstrated its effectiveness against a wide range of pathogenic bacterial and fungal cells with studies suggesting low cytotoxicity towards healthy mammalian cells. However, the antimicrobial mechanism of action of BP is not well understood. Before new applications for this material can be realised, further in-depth investigations are required. In this work, the biochemical interaction between BP and a series of microbial cells is investigated using a variety of microscopy and spectroscopy techniques to provide a greater understanding of the antimicrobial mechanism. Synchrotron macro-attenuated total reflection-Fourier transform infrared (ATR-FTIR) micro-spectroscopy is used to elucidate the chemical changes occurring outside and within the cell of interest after exposure to BP nanoflakes. The ATR-FTIR data, coupled with high-resolution microscopy, reveals major physical and bio-chemical changes to the phospholipids and amide I and II proteins, as well as minor chemical changes to the structural polysaccharides and nucleic acids when compared to untreated cells. These changes can be attributed to the physical interaction of the BP nanoflakes with the cell membranes, combined with the oxidative stress induced by the degradation of the BP nanoflakes. This study provides insight into the biochemical interaction of BP nanoflakes with microbial cells, allowing for a better understanding of the antimicrobial mechanism of action that will be important for the next generation of applications such as implant coatings, wound dressings, or medical surfaces.

Dual-action silver functionalized nanostructured titanium against drug resistant bacterial and fungal species.

HYPOTHESIS: Titanium and its alloys are commonly used implant materials. Once inserted into the body, the interface of the biomaterials is the most likely site for the development of implant-associated infections. Imparting the titanium substrate with high-aspect-ratio nanostructures, which can be uniformly achieved using hydrothermal etching, enables a mechanical contact-killing (mechanoresponsive) mechanism of bacterial and fungal cells. Interaction between cells and the surface shows cellular inactivation via a physical mechanism meaning that careful engineering of the interface is needed to optimse the technology. This mechanism of action is only effective towards surface adsorbed microbes, thus any cells not directly in contact with the substrate will survive and limit the antimicrobial efficacy of the titanium nanostructures. Therefore, we propose that a dual-action mechanoresponsive and chemical-surface approach must be utilised to improve antimicrobial activity. The addition of antimicrobial silver nanoparticles will provide a secondary, chemical mechanism to escalate the microbial response in tandem with the physical puncture of the cells. EXPERIMENTS: Hydrothermal etching is used as a facile method to impart variant nanostrucutres on the titanium substrate to increase the antimicrobial response. Increasing concentrations (0.25 M, 0.50 M, 1.0 M, 2.0 M) of sodium hydroxide etching solution were used to provide differing degrees of nanostructured morphology on the surface after 3 h of heating at 150 °C. This produced titanium nanospikes, nanoblades, and nanowires, respectively, as a function of etchant concentration. These substrates then provided an interface for the deposition of silver nanoparticles via a reduction pathway. Methicillin-resistant Staphylococcous aureus (MRSA) and Candida auris (C. auris) were used as model bacteria and fungi, respectively, to test the effectiveness of the nanostructured titanium with and without silver nanoparticles, and the bio-interactions at the interface. FINDINGS: The presence of nanostructure increased the bactericidal response of titanium against MRSA from âˆ¼ 10 % on commercially pure titanium to a maximum of âˆ¼ 60 % and increased the fungicidal response from âˆ¼ 10 % to âˆ¼ 70 % in C. auris. Introducing silver nanoparticles increased the microbiocidal response to âˆ¼ 99 % towards both bacteria and fungi. Importantly, this study highlights that nanostructure alone is not sufficient to develop a highly antimicrobial titanium substrate. A dual-action, physical and chemical antimicrobial approach is better suited to produce highly effective antibacterial and antifungal surface technologies.

Behavior of Citrate-Capped Ultrasmall Gold Nanoparticles on a Supported Lipid Bilayer Interface at Atomic Resolution.

Nanomaterials have the potential to transform biological and biomedical research, with applications ranging from drug delivery and diagnostics to targeted interference of specific biological processes. Most existing research is aimed at developing nanomaterials for specific tasks such as enhanced biocellular internalization. However, fundamental aspects of the interactions between nanomaterials and biological systems, in particular, membranes, remain poorly understood. In this study, we provide detailed insights into the molecular mechanisms governing the interaction and evolution of one of the most common synthetic nanomaterials in contact with model phospholipid membranes. Using a combination of atomic force microscopy (AFM) and molecular dynamics (MD) simulations, we elucidate the precise mechanisms by which citrate-capped 5 nm gold nanoparticles (AuNPs) interact with supported lipid bilayers (SLBs) of pure fluid (DOPC) and pure gel-phase (DPPC) phospholipids. On fluid-phase DOPC membranes, the AuNPs adsorb and are progressively internalized as the citrate capping of the NPs is displaced by the surrounding lipids. AuNPs also interact with gel-phase DPPC membranes where they partially embed into the outer leaflet, locally disturbing the lipid organization. In both systems, the AuNPs cause holistic perturbations throughout the bilayers. AFM shows that the lateral diffusion of the particles is several orders of magnitude smaller than that of the lipid molecules, which creates some temporary scarring of the membrane surface. Our results reveal how functionalized AuNPs interact with differing biological membranes with mechanisms that could also have implications for cooperative membrane effects with other molecules.

Bottom-up cubosome synthesis without organic solvents.

HYPOTHESIS: Bottom-up synthesis of cubosomes is more energetically favourable than top-down approaches. However, bottom-up methods often rely on organic solvents such as ethanol as diluents, and lead to concurrent formation of liposomes. We propose using non-toxic diluents such as honey, glycerol and lactic acid for bottom-up cubosome synthesis. EXPERIMENTS: Cubosomes were prepared using solutions of phytantriol in a range of diluents including choline chloride-glycerol, honey, lactic acid, glycerol, and ethanol. These solutions were added dropwise to water containing the stabiliser, poloxamer 407, following an established method of cubosome synthesis. The resulting structures were characterised using small-angle X-ray scattering, DLS and cryo-TEM. FINDINGS: Cubosomes were successfully formed using a range of non-toxic diluents. This demonstrates that harmful organic solvents like ethanol are not required, and that the diluents need not be hydrotropes. Furthermore, unlike ethanol, these other diluents allowed formation of cubosomes without concurrent formation of liposomes. Given the huge potential for cubosomes in drug delivery, this new method offers a potentially useful low-cost, low-toxicity synthesis option.