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Proton transportation in proximity to the lipid bilayer membrane surface, where chemical exchange represents a primary pathway, is of significant interest in many applications including cellular energy turnover underlying ATP synthesis, transmembrane mobility, and transport. Lipidic inverse bicontinuous cubic phases (LCPs) are unique membrane structures formed via the spontaneous self-assembly of certain lipids in an aqueous environment. They feature two networks of water channels, separated by a single lipid bilayer which approximates the geometry of a triply periodic minimal surface. When composed of monoolein, the LCP bilayer features two glycerol hydroxyl groups at the lipid–water interface which undergo exchange with water. Depending on the conditions of the aqueous solution used in the formation of LCPs, both resonances of the glycerol hydroxyl groups may be observed by solution 1H NMR. In this study, PFG-NMR and 1D EXSY were employed to gain insight into chemical exchange between the monoolein hydroxyl groups and water in LCPs. Results including the relative population of hydroxyl protons in exchange with water for a number of LCPs at different hydration levels and the exchange rate constants at 35 wt % hydration are reported. Several technical aspects of PFG-NMR and EXSY-NMR for the characterization of chemical exchange in LCPs are discussed, including an alternative way to analyze PFG-NMR data of exchange systems which overcomes the inherent low sensitivity at high diffusion encoding.

Lipidic inverse bicontinuous cubic phases (LCPs), formed via the spontaneous self-assembly of lipids such as monoolein, have found increasing applications in the stabilization and crystallization of integral membrane proteins for structural characterization using X-ray crystallography. Their use as effective drug release matrices has also been demonstrated. Nuclear magnetic resonance (NMR) spectroscopy, both solution and solid state, has previously been employed for the characterization of LCPs and related systems. Herein, we report a number of novel features of solution NMR for probing the fundamental composition and structural properties of monoolein-based LCPs. These include (1) more complete assignments of both 1H and 13C chemical shifts, (2) direct quantification of hydration level in LCPs using one-dimensional (1D) 1H NMR, and (3) monitoring longer-term stability of LCPs and evaluating alterations introduced into standard LCPs at the submolecular level.

Herein, we demonstrate a method for the functionalization of cubic phase lipid nanoparticles (cubosomes) with a series of magnetite (Fe3O4), copper oxide (Cu2O), and silver (Ag) nanocrystals, with prospective applications across a wide range of fields, including antimicrobial treatments. The resulting cubosomes are characterized using small-angle X-ray scattering and dynamic light scattering, demonstrating the retention of a typical cubic phase structure and particle size following nanocrystal encapsulation at concentrations up to 20% w/w. Cryogenic transmission electron microscopy reveals significant loading and association of each nanocrystal type with both monoolein- and phytantriol-based cubosomes. The antibiotic potential of these hybrid nanoparticles is demonstrated for the first time; cubosomes with embedded silver nanocrystals display a high level of antimicrobial activity against both Gram-positive and Gram-negative bacteria, with observed minimum inhibitory concentration values ranging from 15.6–250 μg/mL. Lastly, total internal reflection fluorescence microscopy is used to visualize cubosome–bacteria interactions, suggesting the involvement of particle interactions as a delivery mechanism.

For evolving biological and biomedical applications of hybrid protein?lipid materials, understanding the behavior of the protein within the lipid mesophase is crucial. After more than two decades since the invention of the in meso crystallization method, a protein-eye view of its mechanism is still lacking. Numerous structural studies have suggested that integral membrane proteins preferentially partition at localized flat points on the bilayer surface of the cubic phase with crystal growth occurring from a local fluid lamellar L?phase conduit. However, studies to date have, by necessity, focused on structural transitions occurring in the lipid mesophase. Here, we demonstrate using small-angle neutron scattering that the lipid bilayer of monoolein (the most commonly used lipid for in meso crystallization) can be contrast-matched using deuteration, allowing us to isolate scattering from encapsulated peptides during the crystal growth process for the first time. During in meso crystallization, a clear decrease in form factor scattering intensity of the peptides was observed and directly correlated with crystal growth. A transient fluid lamellar L?phase was observed, providing direct evidence for the proposed mechanism for this technique. This suggests that the peptide passes through a transition from the cubic QIIphase, via an L?phase to the lamellar crystalline Lcphase with similar layered spacing. When high protein loading was possible, the lamellar crystalline Lcphase of the peptide in the single crystals was observed. These findings show the mechanism of in meso crystallization for the first time from the perspective of integral membrane proteins.

The crystallization of oil droplets is critical in the processing and storage of lipid-based food and pharmaceutical products. Arrays of femtoliter droplets on a surface offer a unique opportunity to study surfactant-free colloidlike systems. In this work, the crystal growth process in these confined droplets was followed by cooling a model lipid (trimyristin) from a liquid state utilizing synchrotron small-angle X-ray scattering (SAXS). The measurements by SAXS demonstrated a reduced crystallization rate and a greater degree of supercooling required to trigger lipid crystallization in droplets compared to those of bulk lipids. These results suggest that surface droplets crystallize in a stochastic manner. Interestingly, the crystallization rate is slower for larger femtoliter droplets, which may be explained by the onset of crystallization from the three-phase contact line. The larger surface nanodroplets exhibit a smaller ratio of droplet volume to the length of three-phase contact line and hence a slower crystallization rate.