H-bond network, interfacial tension and chain melting temperature govern phospholipid self-assembly in ionic liquids.

HYPOTHESIS: The self-assembly structures and phase behaviour of phospholipids in protic ionic liquids (ILs) depend on intermolecular forces that can be controlled through changes in the size, polarity, and H-bond capacity of the solvent. EXPERIMENTS: The structure and temperature stability of the self-assembled phases formed by four phospholipids in three ILs was determined by a combination of small- and wide-angle X-ray scattering (SAXS and WAXS) and small-angle neutron scattering (SANS). The phospholipids have identical phosphocholine head groups but different alkyl tail lengths and saturations (DOPC, POPC, DPPC and DSPC), while the ILs' amphiphilicity, H-bond network density and polarity are varied between propylammonium nitrate (PAN) to ethylammonium nitrate (EAN) to ethanolammonium nitrate (EtAN). FINDINGS: The observed structures and phase behaviour of the lipids becomes more surfactant-like with decreasing average solvent polarity, H-bond network density and surface tension. In PAN, all the investigated phospholipids behave like surfactants in water. In EAN they exhibit anomalous phase sequences and unexpected transitions as a function of temperature, while EtAN supports structures that share characteristics with water and EAN. Structures formed are also sensitive to proximity to the lipid chain melting temperature.

How small is the big problem? Small microplastics <300 µm abundant in marine surface waters of the Great Barrier Reef Marine Park.

Particle size plays an important role in determining the behaviour, fate and effects of microplastics (MPs), yet little is known about MPs <300 µm in aquatic environments. Therefore, we performed the first assessment of MPs in marine surface waters around the Whitsunday Islands region of the Great Barrier Reef Marine Park, Australia, to test for the presence of small MPs (50-300 µm) in-situ. Using a modified manta net, we demonstrate that MPs were present in all marine surface water samples, with a mean sea surface concentration of 0.23 ± 0.03 particles m-3. Microplastics were mainly blue, clear and black fibres and fragments, consisting of polyethylene terephthalate, high-density polyethylene and polypropylene plastic polymers. Tourism and marine recreation were considered the major contributing sources of MPs to surface waters around the Whitsunday Islands. Between 10 and 124 times the number of MPs exist in the 50 µm-300 µm size class, compared with the 1 mm-5 mm size range. This finding indicates that the global abundance of small MPs in marine surface waters is grossly underestimated and warrants further investigation. Research into the occurrence, characteristics and environmental fate of MPs <300 µm is needed to improve our understanding of the cumulative threats facing valuable ecosystems due to this smaller, potentially more hazardous size class.

Hydrogen Sulfide-Responsive Bicontinuous Nanospheres.

Block copolymers (BCPs) that can self-assemble into particles and be triggered by disease-specific molecules such as hydrogen sulfide (H2S) have the potential to impact on drug delivery, decreasing off-target toxicities while increasing drug efficacy. However, the incorporation of H2S-responsive aryl azides into BCPs for self-assembly has been limited by heat, light, and radical sensitivities. In this study, a robust activator regenerated by the electron-transfer atom-transfer radical polymerization reaction was used to synthesize aryl-azide-containing BCPs under ambient conditions. Conditions controlling self-assembly of the BCPs into 150-200 nm particles and the physicochemical properties of the particles were investigated. The use of nanoprecipitation with tetrahydrofuran to promote self-assembly of the BCPs resulted in vesicle structures, while dimethylformamide or dimethylsulfoxide resulted in polymeric bicontinuous nanospheres (BCNs). Triggering of the BCPs and particles (vesicles or BCNs) via exposure to H2S revealed that unsubstituted aryl azides were readily reduced (by HS-), resulting in particle disruption or cross-linking. The relative polar nature of the particle bilayers containing unsubstituted aryl azides and the open structure of the BCNs did however limit encapsulation of small hydrophilic and hydrophobic payloads. Incorporation of a benzylamide substituent onto the aryl azide group increased the hydrophobicity of the particles and encapsulation of hydrophilic cargo but reduced sensitivity to H2S, likely due to the reduced penetration of HS- into the bilayer.

Synthesis of 2D Porous Crystalline Materials in Simulated Microgravity.

To date, crystallization studies conducted in space laboratories, which are prohibitively costly and unsuitable to most research laboratories, have shown the valuable effects of microgravity during crystal growth and morphogenesis. Herein, an easy and highly efficient method is shown to achieve space-like experimentation conditions on Earth employing custom-made microfluidic devices to fabricate 2D porous crystalline molecular frameworks. It is confirmed that experimentation under these simulated microgravity conditions has unprecedented effects on the orientation, compactness and crack-free generation of 2D porous crystalline molecular frameworks as well as in their integration and crystal morphogenesis. It is believed that this work will provide a new "playground" to chemists, physicists, and materials scientists that desire to process unprecedented 2D functional materials and devices.

Enzymatic hydrolysis of monoacylglycerols and their cyclopropanated derivatives: Molecular structure and nanostructure determine the rate of digestion.

Colloidal lipidic particles with different space groups and geometries (mesosomes) are employed in the development of new nanosystems for the oral delivery of drugs and nutrients. Understanding of the enzymatic digestion rate of these particles is key to the development of novel formulations. In this work, the molecular structure of the lipids has been systematically tuned to examine the effect on their self-assembly and digestion rate. The kinetic and phase changes during the lipase-catalysed hydrolysis of mesosomes formed by four synthetic cyclopropanated lipids and their cis-unsaturated analogues were monitored by dynamic small angle X-ray scattering and acid/base titration. It was established that both the phase behaviour and kinetics of the hydrolysis are greatly affected by small changes in the molecular structure of the lipid as well as by the internal nanostructure of the colloidal particles.

Lipid-based mesophases as matrices for nanoscale reactions.

Lipidic mesophases are versatile bioorganic materials that have been effectively employed as nanoscale matrices for membrane protein crystallization, drug delivery and as food emulsifiers over the last 30 years. In this review, the focus is upon studies that have employed non-lamellar lipid mesophases as matrices for organic, inorganic and enzymatic reactions. The ability of lipidic mesophases to incorporate hydrophilic, amphiphilic and hydrophobic molecules, together with the high interfacial area of the lipidic cubic and inverse hexagonal phases has been exploited in heterogeneous catalysis as well as for enzyme immobilization. The unique nanostructure of these mesophases is the driving force behind their ability to act as templates for synthesis, resulting in the creation of highly ordered polymeric and inorganic materials with complex geometries.

Understanding the assembly of amphiphilic additives in bulk and dispersed non-lamellar lipid-based matrices: Phosphorylation, H-bonding and ionisation.

The aqueous channel size of lipidic cubic phases can be a limiting factor for certain applications. For this reason, additives have been used to exquisitely control their nanostructure. In this study, two families of primary phosphoesters have been designed, synthesised and utilised to determine the effect of the positioning of the guest additive at the interface of the host mesophase, and to contrast the effect of headgroup ionisation and protonation. A general methodology has been developed to produce primary phosphoesters, and a unique use of 31P NMR has been used in order to systematically investigate the influence of these additives on monoolein- and phytantriol-based bulk lipidic cubic phases and dispersed cubosomes. In general, di-phosphorylated additives exhibit a greater effect upon lipid packing than the mono- and tri-phosphorylated molecules due to their optimal positioning. In dispersion, the protonation state of the phosphate headgroups was manipulated by altering the pH, where shifts in pKa determined by 31P NMR were used as a fluorescent label-free method to identify the location and ionisation state of the phosphate additives. This study systematically evaluates the influence of the positioning of the additive, headgroup size and charge of phosphorylated lipids on the behaviour of lipidic mesophases.

Structural Transformation in Vesicles upon Hydrolysis of Phosphatidylethanolamine and Phosphatidylcholine with Phospholipase C.

This study provides insights into dynamic nanostructural changes in phospholipid systems during hydrolysis with phospholipase C, the fate of the hydrolysis products, and the kinetics of lipolysis. The effect of lipid restructuring of the vesicle was investigated using small-angle X-ray scattering and cryogenic scanning electron microscopy. The rate and extent of phospholipid hydrolysis were quantified using nuclear magnetic resonance. Hydrolysis of two phospholipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), results in the cleavage of the molecular headgroup, causing two strikingly different changes in lipid self-assembly. The diacylglycerol product of PC escapes the lipid bilayer, whereas the diacylglycerol product adopts a different configuration within the lipid bilayer of the PE vesicles. These results are then discussed concerning the change of the lipid configuration upon the lipid membrane and its potential implications in vivo, which is of significant importance for the detailed understanding of the fate of lipidic particles and the rational design of enzyme-responsive lipid-based drug delivery systems.

Hexosomes with Undecylenic Acid Efficient against Candida albicans.

Due to the growing issues with fungal infections, especially with Candida, there is still a need to develop novel anti-Candida materials. One of the known antifungal agents is undecylenic acid (UA), which still cannot be efficiently used due to its oily nature, and thus limited solubility. By taking advantage of the properties of UA, we developed an emulsion with hexagonal phase, i.e., hexosomes, whose structure and morphology was studied by small-angle X-ray scattering and cryo-electron microscopy, respectively. The presence of UA in the hexosome was confirmed by spectroscopy. Moreover, we studied the anti-Candida effect of hexosomes and their cytotoxicity toward human cells. The minimal inhibitory concentration for the 50% and 90% Candida-growth reduction was found at 0.01 and 0.16 wt % hexosomes, respectively (i.e., 2 and 32 pghex/C.a.cell, respectively). The percentage of metabolically active Candida was reduced by 72-96% at hexosome concentrations of 1.0-8.2 pghex/C.a.cell as compared to untreated Candida. Furthermore, at the same concentration range the embedded filamentation test after 24 and 48 h showed the inhibition of both the filamentation and growth of Candida, while the preliminary toxicity test showed that hexosomes were nontoxic for human cells. All these render the here-developed hexosomes with UA efficient and promising anti-Candida agents.

Ice-Templated and Cross-Linked Amyloid Fibril Aerogel Scaffolds for Cell Growth.

Amyloid fibrils prepared from ß-lactoglobulin were used to form freeze-dried and cross-linked aerogels. By varying the fibril concentration and freezing gradient, it was possible to control the pore structure and elastic modulus of the aerogels within one order of magnitude from ∼20 to ∼200 kPa. Using butane tetracarboxylic acid as cross-linker, these aerogels maintained their monolithic shape under aqueous conditions, displaying elastic behavior and a modulus in the range of ∼4-40 kPa. When explored as scaffolds for cell growth, the amyloid fibril aerogels demonstrated biocompatibility and led to the successful penetration and permeation of two epithelial cell lines (Caco-2 and HT29) throughout the scaffold. These soft, elastic, and water-stable biomaterials expand the scope of amyloid fibril aerogels, making them suitable for wet-state applications such as heterogeneous catalysis, purification membranes, and 3D matrices for cell growth.

Investigation of Donor-Acceptor Stenhouse Adducts as New Visible Wavelength-Responsive Switching Elements for Lipid-Based Liquid Crystalline Systems.

The ability of donor-acceptor Stenhouse adducts (DASAs) to function as a green light responsive switch for lipid-based liquid crystalline drug delivery systems was investigated. The host matrix comprising phytantriol cubic phase was selected due to its high sensitivity toward changes in lipid packing. Small-angle X-ray scattering demonstrated that the matrix undergoes rapid and fully reversible order-order phase transitions upon irradiation with 532 nm light, converting between the bicontinuous cubic phase and reversed hexagonal phases. This approach shows promise for use as an actuator for the development of visible wavelength light-activated, "on-demand" drug delivery systems.

Responsive self-assembled nanostructured lipid systems for drug delivery and diagnostics.

While stimuli-responsive polymers have received a huge amount of attention in the literature, responsive lipid-based mesophase systems offer unique opportunities in biomedical applications such as drug delivery and biosensing. The different mesophase equilibrium structures enables dynamic switching between nanostructures to facilitate drug release or as a transducer for recognition events. In drug delivery, this behavior offers researchers the means to deliver a therapeutic payload at a specific rate and time i.e. 'on-demand'. This review summarizes the distinctive features of these multifaceted materials and aggregates the current state of the art research from our groups and others into the use of these materials as bulk gels and nanostructured dispersions for drug delivery, biosensing and diagnostics.

Lipid-based drug delivery systems in the treatment of wet age-related macular degeneration.

Recent advances in drug delivery technology have amplified potential opportunities to treat the debilitating diseases that affect the posterior segment of the eye in a less invasive and more efficient manner. Current methods for effective drug delivery to the back of the eye are hindered by many barriers and limitations. As a consequence, considerable efforts have been directed towards developing new materials to selectively deliver drug directly to the target site. This review focuses on lipid-based delivery systems which show promise in improving treatment for the most common disease of the posterior segment of the eye in the developed world, age-related macular degeneration, with an emphasis upon on-demand delivery systems as they have greater potential to overcome the current limitations.

Lyotropic Liquid Crystalline Cubic Phases as Versatile Host Matrices for Membrane-Bound Enzymes.

Lyotropic liquid crystalline cubic mesophases can function as host matrices for enzymes because of their biomimetic structural characteristics, optical transparency, and capability to coexist with water. This study demonstrates that the in meso immobilized membrane-bound enzyme d-fructose dehydrogenase (FDH) preserves its full activity, follows ideal Michaelis-Menten kinetics, and shows improved stability compared to its behavior in solution. Even after 5 days, the immobilized FDH retained its full activity in meso, whereas a model hydrophilic enzyme, horseradish peroxidase, maintained only 21% of its original activity. We reason that the lipidic bilayers in the three-dimensional structures of cubic mesophases provide an ideal environment for the reconstitution of a membrane-bound enzyme. The preserved activity, long-term stability, and reusability demonstrate that these hybrid nanomaterials are ideal matrices for biosensing and biocatalytic fuel cell applications.

External manipulation of nanostructure in photoresponsive lipid depot matrix to control and predict drug release in vivo.

On-demand drug delivery systems are highly promising to control the time-course of drug release and ultimately optimize drug concentration time profiles in patients. Lipid based lyotropic liquid crystalline mesophases have demonstrated exceptional responsiveness to external stimuli such as heat, pH and light. Our objective was to quantitatively characterize the time-course of light activated drug release from near infrared (NIR) activated photothermal systems using ex vivo and in vivo studies. Photoresponsive hybrid gold nanorod-liquid crystalline matrices were prepared and loaded into custom-made implants which were inserted into subcutaneous tissues in rats. Time resolved SAXS studies showed the abdomen to be the best site of implantation to achieve in vivo activation of the subcutaneous dose from by the NIR laser. External control of drug release was achieved via NIR laser light and plasma concentrations of the model drug were determined over time. Laser activation achieved a phase change of the photoresponsive formulations and thereby a considerable change in the rate of drug release. Population pharmacokinetic modeling of all results simultaneously revealed a two stage release process unique to these liquid crystalline matrices. The developed structural model was able to successfully describe also the results of our previous study in 2009 where a change in temperature was utilized to trigger subcutaneous drug release. Thus, modeling of the data proved to be a valuable analytical tool which provided a quantitative understanding of the time-course of drug release in vivo and will be essential in the development of these matrices as on-demand release systems.

Phospholipid-based self-assembled mesophase systems for light-activated drug delivery.

The manipulation of the structure of phospholipid-based mesophases to induce a slow to fast drug release profile has potential for use in therapeutic situations where continuous absorption of drug is not desirable and reduce the frequency of injection for short acting or rapidly cleared drugs in treatments for diseases such as macular degeneration. This study had two aims; firstly to confirm the phase behaviour of 20 mol% cholesterol in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), which was previously reported to transition from lamellar (slow release) to bicontinuous cubic (fast release) phase with increasing temperature. Contrary to the literature, no bicontinuous cubic phase was observed but a transition to the inverse hexagonal phase occurred at all POPE : cholesterol ratios investigated. The second aim was to render these mesophases responsive to near-infrared laser (NIR) irradiation by incorporation of gold nanorods (GNR) incorporated into the POPE system to induce photothermal heating. The inclusion of 3 nM GNR in POPE systems induced reversible disruption of lipid packing equivalent to increasing the temperature to 55 °C when irradiated for 30 s. This study confirmed that although the previously published phase behavior was not correct, GNR and NIR can be used to manipulate the self-assembled mesophases in phospholipid-based systems and highlights the potential for a phospholipid-based light-activated drug delivery system.

pH-responsive lyotropic liquid crystals and their potential therapeutic role in cancer treatment.

A weak amphiphilic base, pyridinylmethyl linoleate, is blended with monolinolein, yielding mesophases with a pH-induced hexagonal-to-cubic transition at pH ≤ 5.5. We show the potential therapeutic role of this mesophase in treating cancerous tissues exploiting their more acidic pH compared to healthy tissues. In vitro release studies with doxorubicin on HT29 human colon cancer cells show a 10-fold faster release and 3-fold increased efficiency for killing cancer cells at pH 5.5 versus pH 7.4, demonstrating the potential of this strategy in cancer treatment.

Oil transfer converts phosphatidylcholine vesicles into nonlamellar lyotropic liquid crystalline particles.

There is a need for the development of low-energy dispersion methods tailored to the formation of phospholipid-based nonlamellar lyotropic liquid crystalline (LLC) particles for delivery system applications. Here, facile formation of nonlamellar LLC particles was obtained by simple mixing of a phosphatidylcholine (PC) liposome solution and an oil-in-water emulsion, with limonene or isooctane as an oil. The internal structure of the particles was controlled by the PC-to-oil ratio, consistently with the sequence observed in bulk phase. For the first time, reverse micellar cubosomes with Fm3̅m inner structure were produced. The size, morphology, and inner structure of the particles were characterized by small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and freeze-fracture cryo scanning electron microscopy (cryo-SEM). These findings pave the way to new strategies in low-energy formulation of LLC delivery systems.

Understanding the photothermal heating effect in non-lamellar liquid crystalline systems, and the design of new mixed lipid systems for photothermal on-demand drug delivery.

Lipid-based liquid crystalline systems are showing potential as stimuli-responsive nanomaterials, and NIR-responsive gold nanoparticles have been demonstrated to provide control of transitions in non-lamellar phases. In this study, we focus on a deeper understanding of the photothermal response of both lamellar and non-lamellar phases, and new systems formed by alternative lipid systems not previously reported, by linking the photothermal heating to the bulk thermal properties of the materials. Dynamic photothermal studies were performed using NIR laser irradiation and monitoring the structural response using time resolved small angle X-ray scattering for the bulk phases and hexosomes. In addition, cryoFESEM and cryoTEM were used to visualise and assess the effect of GNR incorporation into hexagonal phase nanostructures. The ability of the systems to respond to photothermal heating was correlated with the thermal phase behaviour and heat capacities of the different structures. Access to alternative phase transitions in these systems and understanding the likely photothermal response will facilitate different modes of application of these hybrid nanomaterials for on-demand drug delivery applications.

Generation of geometrically ordered lipid-based liquid-crystalline nanoparticles using biologically relevant enzymatic processing.

High-symmetry lipid nanoparticles with internal bicontinuous cubic phase structure (cubosomes) are prepared from a simple emulsion containing a mixture of a nondigestible lipid (phytantriol) and a digestible short-chained triglyceride using enzymatic lipolysis of the incorporated short-chained triglyceride. The lipolytic products partition away from the nondigestible lipid, resulting in crystallization of the cubic-phase internal structure. Time-resolved small-angle X-ray scattering revealed the kinetics of the disorder-to-order transition, with cryo-transmission electron microscopy showing an absence of liposomes. The new approach offers a new "sideways" method for the generation of lipid-based nanostructured materials that avoids the problems of top-down and bottom-up approaches.