Quantitative Analysis of 2D EXSY NMR Spectra of Strongly Coupled Spin Systems in Transmembrane Exchange.

Solute translocation by membrane transport proteins is a vital biological process that can be tracked, on the sub-second timescale, using nuclear magnetic resonance (NMR). Fluorinated substrate analogues facilitate such studies because of high sensitivity of 19 F NMR and absence of background signals. Accurate extraction of translocation rate constants requires precise quantification of NMR signal intensities. This becomes complicated in the presence of J-couplings, cross-correlations, and nuclear Overhauser effects (NOE) that alter signal integrals through mechanisms unrelated to translocation. Geminal difluorinated motifs introduce strong and hard-to-quantify contributions from non-exchange effects, the nuanced nature of which makes them hard to integrate into data analysis methodologies. With analytical expressions not being available, numerical least squares fitting of theoretical models to 2D spectra emerges as the preferred quantification approach. For large spin systems with simultaneous coherent evolution, cross-relaxation, cross-correlation, conformational exchange, and membrane translocation between compartments with different viscosities, the only available simulation framework is Spinach. In this study, we demonstrate GLUT-1 dependent membrane transport of two model sugars featuring CF2 and CF2 CF2 fluorination motifs, with precise determination of translocation rate constants enabled by numerical fitting of 2D EXSY spectra. For spin systems and kinetic networks of this complexity, this was not previously tractable.

High-aspect-ratio dielectric pillar with nanocavity backed by metal substrate in the infrared range.

We investigated absorption and field enhancements of shallow nanocavities on top of high-aspect-ratio dielectric pillars in the infrared range. The structure includes a high-aspect-ratio nanopillar array of high refractive index, with nano-cavities on top of the pillars, and a metal plane at the bottom. The enhancement factor of electric field intensity reaches 3180 in the nanocavities and peak absorption reaches 99%. We also investigated the finite-size effect of the presented structure to simulate real experiments. Due to its narrow absorption bandwidth 3.5 nm, it can work as a refractive index sensor with sensitivity 297.5 nm/RIU and figure of merit 85. This paves the way to directly control light field at the nanoscales in the infrared light range. The investigated nanostructure will find applications in multifunctional photonics devices such as chips for culturing cells, refractive index sensors, biosensors of single molecule detection and nonlinear sensors.

A maladaptive feedback mechanism between the extracellular matrix and cytoskeleton contributes to hypertrophic cardiomyopathy pathophysiology.

Hypertrophic cardiomyopathy is an inherited disorder due to mutations in contractile proteins that results in a stiff, hypercontractile myocardium. To understand the role of cardiac stiffness in disease progression, here we create an in vitro model of hypertrophic cardiomyopathy utilizing hydrogel technology. Culturing wild-type cardiac myocytes on hydrogels with a Young's Moduli (stiffness) mimicking hypertrophic cardiomyopathy myocardium is sufficient to induce a hypermetabolic mitochondrial state versus myocytes plated on hydrogels simulating healthy myocardium. Significantly, these data mirror that of myocytes isolated from a murine model of human hypertrophic cardiomyopathy (cTnI-G203S). Conversely, cTnI-G203S myocyte mitochondrial function is completely restored when plated on hydrogels mimicking healthy myocardium. We identify a mechanosensing feedback mechanism between the extracellular matrix and cytoskeletal network that regulates mitochondrial function under healthy conditions, but participates in the progression of hypertrophic cardiomyopathy pathophysiology resulting from sarcomeric gene mutations. Importantly, we pinpoint key 'linker' sites in this schema that may represent potential therapeutic targets.

Molecular interactions of STAC proteins with skeletal muscle dihydropyridine receptor and excitation-contraction coupling.

Excitation-contraction coupling (ECC) is the physiological process in which an electrical signal originating from the central nervous system is converted into muscle contraction. In skeletal muscle tissue, the key step in the molecular mechanism of ECC initiated by the muscle action potential is the cooperation between two Ca2+ channels, dihydropyridine receptor (DHPR; voltage-dependent L-type calcium channel) and ryanodine receptor 1 (RyR1). These two channels were originally postulated to communicate with each other via direct mechanical interactions; however, the molecular details of this cooperation have remained ambiguous. Recently, it has been proposed that one or more supporting proteins are in fact required for communication of DHPR with RyR1 during the ECC process. One such protein that is increasingly believed to play a role in this interaction is the SH3 and cysteine-rich domain-containing protein 3 (STAC3), which has been proposed to bind a cytosolic portion of the DHPR α1S subunit known as the II-III loop. In this work, we present direct evidence for an interaction between a small peptide sequence of the II-III loop and several residues within the SH3 domains of STAC3 as well as the neuronal isoform STAC2. Differences in this interaction between STAC3 and STAC2 suggest that STAC3 possesses distinct biophysical features that are potentially important for its physiological interactions with the II-III loop. Therefore, this work demonstrates an isoform-specific interaction between STAC3 and the II-III loop of DHPR and provides novel insights into a putative molecular mechanism behind this association in the skeletal muscle ECC process.

Surface model of the human red blood cell simulating changes in membrane curvature under strain.

We present mathematical simulations of shapes of red blood cells (RBCs) and their cytoskeleton when they are subjected to linear strain. The cell surface is described by a previously reported quartic equation in three dimensional (3D) Cartesian space. Using recently available functions in Mathematica to triangularize the surfaces we computed four types of curvature of the membrane. We also mapped changes in mesh-triangle area and curvatures as the RBCs were distorted. The highly deformable red blood cell (erythrocyte; RBC) responds to mechanically imposed shape changes with enhanced glycolytic flux and cation transport. Such morphological changes are produced experimentally by suspending the cells in a gelatin gel, which is then elongated or compressed in a custom apparatus inside an NMR spectrometer. A key observation is the extent to which the maximum and minimum Principal Curvatures are localized symmetrically in patches at the poles or equators and distributed in rings around the main axis of the strained RBC. Changes on the nanometre to micro-meter scale of curvature, suggest activation of only a subset of the intrinsic mechanosensitive cation channels, Piezo1, during experiments carried out with controlled distortions, which persist for many hours. This finding is relevant to a proposal for non-uniform distribution of Piezo1 molecules around the RBC membrane. However, if the curvature that gates Piezo1 is at a very fine length scale, then membrane tension will determine local curvature; so, curvatures as computed here (in contrast to much finer surface irregularities) may not influence Piezo1 activity. Nevertheless, our analytical methods can be extended address these new mechanistic proposals. The geometrical reorganization of the simulated cytoskeleton informs ideas about the mechanism of concerted metabolic and cation-flux responses of the RBC to mechanically imposed shape changes.

Enhanced Ca2+ influx in mechanically distorted erythrocytes measured with 19F nuclear magnetic resonance spectroscopy.

We present the first direct nuclear magnetic resonance (NMR) evidence of enhanced entry of Ca2+ ions into human erythrocytes (red blood cells; RBCs), when these cells are mechanically distorted. For this we loaded the RBCs with the fluorinated Ca2+ chelator, 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA), and recorded 19F NMR spectra. The RBCs were suspended in gelatin gel in a special stretching/compression apparatus. The 5FBAPTA was loaded into the cells as the tetraacetoxymethyl ester; and 13C NMR spectroscopy with [1,6-13C]D-glucose as substrate showed active glycolysis albeit at a reduced rate in cell suspensions and gels. The enhancement of Ca2+ influx is concluded to be via the mechanosensitive cation channel Piezo1. The increased rate of influx brought about by the activator of Piezo1, 2-[5-[[(2,6-dichlorophenyl)methyl]thio]-1,3,4-thiadiazol-2-yl]-pyrazine (Yoda1) supported this conclusion; while the specificity of the cation-sensing by 5FBAPTA was confirmed by using the Ca2+ ionophore, A23187.

Sensing refractive index gradients along dielectric nanopillar metasurfaces.

Metasurfaces exhibit unique optical properties that depend on the ratio of their refractive index and that of their surroundings. As such, they are effective for sensing global changes in refractive index based on the shifts of resonances in their reflectivity spectra. However, when used as a biosensor, the metasurface can be exposed to a spatial distribution of biomolecules that brings about gradients in refractive index along the plane of the metasurface. Such gradients produce complex global reflectivity spectrum but with distinct optical enhancements in localized areas along the metasurface. Here, we propose a unique sensing paradigm that images and maps out the optical enhancements that are correlated with the spatial distribution of the refractive index. Moreover, we designed a metasurface whose resonances can be tuned to detect a range of refractive indices. Our metasurface consists of silicon nanopillars with a cylindrical nanotrench at their centers and a metal plane at the base. To assess its feasibility, we performed numerical simulations to show that the design effectively produces the desired reflectivity spectrum with resonances in the near-infrared. Using an incident light tuned to one of its resonances, our simulations further show that the field enhancements are correlated with the spatial mapping of the gradients of refractive indices along the metasurface.

Excitation-contraction coupling in skeletal muscle: recent progress and unanswered questions.

Excitation-contraction coupling (ECC) is a physiological process that links excitation of muscles by the nervous system to their mechanical contraction. In skeletal muscle, ECC is initiated with an action potential, generated by the somatic nervous system, which causes a depolarisation of the muscle fibre membrane (sarcolemma). This leads to a rapid change in the transmembrane potential, which is detected by the voltage-gated Ca2+ channel dihydropyridine receptor (DHPR) embedded in the sarcolemma. DHPR transmits the contractile signal to another Ca2+ channel, ryanodine receptor (RyR1), embedded in the membrane of the sarcoplasmic reticulum (SR), which releases a large amount of Ca2+ ions from the SR that initiate muscle contraction. Despite the fundamental role of ECC in skeletal muscle function of all vertebrate species, the molecular mechanism underpinning the communication between the two key proteins involved in the process (DHPR and RyR1) is still largely unknown. The goal of this work is to review the recent progress in our understanding of ECC in skeletal muscle from the point of view of the structure and interactions of proteins involved in the process, and to highlight the unanswered questions in the field.

Rapid zero-trans kinetics of Cs+ exchange in human erythrocytes quantified by dissolution hyperpolarized 133Cs+ NMR spectroscopy.

Transmembrane flux of Cs+ (a K+ congener) was measured in human red blood cells (RBCs; erythrocytes) on the 10-s time scale. This is the first report on dissolution dynamic nuclear polarization (dDNP) nuclear magnetic resonance (NMR) spectroscopy with this nuclide in mammalian cells. Four technical developments regularized sample delivery and led to high quality NMR spectra. Cation-free media with the Piezo1 (mechanosensitive cation channel) activator yoda1 maximized the extent of membrane transport. First-order rate constants describing the fluxes were estimated using a combination of statistical methods in Mathematica, including the Markov chain Monte Carlo (MCMC) algorithm. Fluxes were in the range 4-70 µmol Cs+ (L RBC)-1 s-1; these are smaller than for urea, but comparable to glucose. Methodology and analytical procedures developed will be applicable to transmembrane cation transport studies in the presence of additional Piezo1 effectors, to other cellular systems, and potentially in vivo.

Glyoxalase activity in human erythrocytes and mouse lymphoma, liver and brain probed with hyperpolarized 13C-methylglyoxal.

Methylglyoxal is a faulty metabolite. It is a ubiquitous by-product of glucose and amino acid metabolism that spontaneously reacts with proximal amino groups in proteins and nucleic acids, leading to impairment of their function. The glyoxalase pathway evolved early in phylogeny to bring about rapid catabolism of methylglyoxal, and an understanding of the role of methylglyoxal and the glyoxalases in many diseases is beginning to emerge. Metabolic processing of methylglyoxal is very rapid in vivo and thus notoriously difficult to detect and quantify. Here we show that 13C nuclei in labeled methylglyoxal can be hyperpolarized using dynamic nuclear polarization, providing 13C nuclear magnetic resonance signal enhancements in the solution state close to 5,000-fold. We demonstrate the applications of this probe of metabolism for kinetic characterization of the glyoxalase system in isolated cells as well as mouse brain, liver and lymphoma in vivo.

Transmembrane Exchange of Fluorosugars: Characterization of Red Cell GLUT1 Kinetics Using 19F NMR.

We have developed a new approach, to our knowledge, to quantify the equilibrium exchange kinetics of carrier-mediated transmembrane transport of fluorinated substrates. The method is based on adapted kinetic theory that describes the concentration dependence of the transmembrane exchange rates of two competing, simultaneously transported species. Using the new approach, we quantified the kinetics of membrane transport of both anomers of three monofluorinated glucose analogs in human erythrocytes (red blood cells) using 19F NMR exchange spectroscopy. An inosine-based glucose-free medium was shown to promote survival and stable metabolism of red blood cells over the duration of the experiments (several hours). Earlier NMR studies only yielded the apparent rate constants and transmembrane fluxes of the anomeric species, whereas we could categorize the two anomers in terms of the catalytic activity (specificity constants) of the glucose transport protein GLUT1 toward them. Differences in the membrane permeability of the three glucose analogs were qualitatively interpreted in terms of local perturbations in the bonding of substrates to key amino acid residues in the active site of GLUT1. The methodology of this work will be applicable to studies of other carrier-mediated membrane transport processes, especially those with competition between simultaneously transported species. The GLUT1-specific results can be applied to the design of probes of glucose transport or inhibitors of glucose metabolism in cells, including those exhibiting the Warburg effect.

1H, 13C and 15N backbone NMR chemical shift assignments of the C-terminal P4 domain of Ahnak.

Ahnak is a ~ 700 kDa polypeptide that was originally identified as a tumour-related nuclear phosphoprotein, but later recognized to play a variety of diverse physiological roles related to cell architecture and migration. A critical function of Ahnak is modulation of Ca2+ signaling in cardiomyocytes by interacting with the ß subunit of the L-type Ca2+ channel (CaV1.2). Previous studies have identified the C-terminal region of Ahnak, designated as P3 and P4 domains, as a key mediator of its functional activity. We report here the nearly complete 1H, 13C and 15N backbone NMR chemical shift assignments of the 11 kDa C-terminal P4 domain of Ahnak. This study lays the foundations for future investigations of functional dynamics, structure determination and interaction site mapping of the CaV1.2-Ahnak complex.

The NMR 'split peak effect' in cell suspensions: Historical perspective, explanation and applications.

The physicochemical environment inside cells is distinctly different from that immediately outside. The selective exchange of ions, water and other molecules across the cell membrane, mediated by integral, membrane-embedded proteins is a hallmark of living systems. There are various methodologies available to measure the selectivity and rates (kinetics) of such exchange processes, including several that take advantage of the non-invasive nature of NMR spectroscopy. A number of solutes, including particular inorganic ions, show distinctive NMR behaviour, in which separate resonances arise from the intra- and extracellular solute populations, without the addition of shift reagents, differences in pH, or selective binding partners. This 'split peak effect/phenomenon', discovered in 1984, has become a valuable tool, used in many NMR studies of cellular behaviour and function. The explanation for the phenomenon, based on the differential hydrogen bonding of the reporter solutes to water, and the various ways in which this phenomenon has been used to investigate aspects of cellular biochemistry and physiology, are the topics of this review.

Sub-minute kinetics of human red cell fumarase: 1 H spin-echo NMR spectroscopy and 13 C rapid-dissolution dynamic nuclear polarization.

Fumarate is an important probe of metabolism in hyperpolarized magnetic resonance imaging and spectroscopy. It is used to detect the release of fumarase in cancer tissues, which is associated with necrosis and drug treatment. Nevertheless, there are limited reports describing the detailed kinetic studies of this enzyme in various cells and tissues. Thus, we aimed to evaluate the sub-minute kinetics of human red blood cell fumarase using nuclear magnetic resonance (NMR) spectroscopy, and to provide a quantitative description of the enzyme that is relevant to the use of fumarate as a probe of cell rupture. The fumarase reaction was studied using time courses of 1 H spin-echo and 13 C-NMR spectra. 1 H-NMR experiments showed that the fumarase reaction in hemolysates is sufficiently rapid to make its kinetics amenable to study in a period of approximately 3 min, a timescale characteristic of hyperpolarized 13 C-NMR spectroscopy. The rapid-dissolution dynamic nuclear polarization (RD-DNP) technique was used to hyperpolarize [1,4-13 C]fumarate, which was injected into concentrated hemolysates. The kinetic data were analyzed using recently developed FmRα analysis and modeling of the enzymatic reaction using Michaelis-Menten equations. In RD-DNP experiments, the decline in the 13 C-NMR signal from fumarate, and the concurrent rise and fall of that from malate, were captured with high spectral resolution and signal-to-noise ratio, which allowed the robust quantification of fumarase kinetics. The kinetic parameters obtained indicate the potential contribution of hemolysis to the overall rate of the fumarase reaction when 13 C-NMR RD-DNP is used to detect necrosis in animal models of implanted tumors. The analytical procedures developed will be applicable to studies of other rapid enzymatic reactions using conventional and hyperpolarized substrate NMR spectroscopy.

Accelerating metabolism and transmembrane cation flux by distorting red blood cells.

Under static conditions, mammalian red blood cells (RBCs) require a continuous supply of energy, typically via glucose, to maintain their biconcave disc shape. Mechanical distortion, in a complementary way, should lead to increased energy demand that is manifest in accelerated glycolysis. The experimental challenge in observing this phenomenon was met by reversibly and reproducibly distorting the cells and noninvasively measuring glycolytic flux. This was done with a gel-distorting device that was coupled with 13C nuclear magnetic resonance (NMR) spectroscopy. We measured [3-13C]l-lactate production from [1,6-13C]d-glucose in the RBCs suspended in gelatin gels, and up to 90% rate enhancements were recorded. Thus, for the first time, we present experiments that demonstrate the linkage of mechanical distortion to metabolic changes in whole mammalian cells. In seeking a mechanism for the linkage between shape and energy supply, we measured transmembrane cation flux with Cs+ (as a K+ congener) using 133Cs NMR spectroscopy, and the cation flux was increased up to fivefold. The postulated mechanism for these notable (in terms of whole-body energy consumption) responses is stimulation of Ca-adenosine triphosphatase by increased transmembrane flux of Ca2+ via the channel protein Piezo1 and increased glycolysis because its flux is adenosine triphosphate demand-regulated.

Hypoxia-Responsive Cobalt Complexes in Tumor Spheroids: Laser Ablation Inductively Coupled Plasma Mass Spectrometry and Magnetic Resonance Imaging Studies.

Dense tumors are resistant to conventional chemotherapies due to the unique tumor microenvironment characterized by hypoxic regions that promote cellular dormancy. Bioreductive drugs that are activated in response to this hypoxic environment are an attractive strategy for therapy with anticipated lower harmful side effects in normoxic healthy tissue. Cobalt bioreductive pro-drugs that selectively release toxic payloads upon reduction in hypoxic cells have shown great promise as anticancer agents. However, the bioreductive response in the tumor microenvironment must be better understood, as current techniques for monitoring bioreduction to Co(II) such as X-ray absorption near-edge structure and extended X-ray absorption fine structure provide limited information on speciation and require synchrotron radiation sources. Here, we present magnetic resonance imaging (MRI) as an accessible and powerful technique to monitor bioreduction by treating the cobalt complex as an MRI contrast agent and monitoring the change in water signal induced by reduction from diamagnetic Co(III) to paramagnetic Co(II). Cobalt pro-drugs built upon the tris(2-pyridylmethyl)amine ligand scaffold with varying charge were investigated for distribution and activity in a 3D tumor spheroid model by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and MRI. In addition, paramagnetic 1H NMR spectroscopy of spheroids enabled determination of the speciation of activated Co(II)TPAx complexes. This study demonstrates the utility of MRI and associated spectroscopy techniques for understanding bioreductive cobalt pro-drugs in the tumor microenvironment and has broader implications for monitoring paramagnetic metal-based therapies.

Bile salt stimulated lipase: Inhibition by phospholipids and relief by phospholipase A2.

INTRODUCTION: Bile salt stimulated lipase (BSSL; Enzyme Commission (EC) number 3.1.1.13) has been a candidate triglyceridase for improving enzyme therapy for pancreatic insufficiency; however, its efficacy is near absent. We hypothesise that similarly to pancreatic lipase, BSSL is inhibited by phospholipids and this inhibition is relieved by Phospholipase A2 (PLA2; EC 3.1.1.4), and the present study was undertaken to explore this possibility. MATERIALS AND METHODS: Synthetic emulsions of triglyceride and phosphatidylcholine (PC) or lysophosphatidylcholine (LPC)/bile salt mixed micelles were used as a model of intestinal digestion-media. The effect of PLA2 treatment of systems containing PC on BSSL activity was also explored. Automatic titration at constant pH (pH-stat) and nuclear magnetic resonance (NMR) spectroscopy were used to measure the rate and identify products of lipolysis. RESULTS: PC was inhibitory to BSSL activity, while LPC became inhibitory only above an LPC/bile salt concentration ratio of 0.3. PLA2 treatment relieved the inhibition only below this ratio, despite its complete phospholipid-hydrolysing action. Thus, LPC had an inhibitory effect at higher concentrations. CONCLUSIONS: These results may implicate a change in the design of enzyme therapy in patients with pancreatic exocrine insufficiency. Supplementation of BSSL with PLA2 could improve patient health with adequate manipulation of phospholipid and lysophospholipid concentrations in the intestinal fluid.

The S1 helix critically regulates the finely tuned gating of Kv11.1 channels.

Congenital mutations in the cardiac Kv11.1 channel can cause long QT syndrome type 2 (LQTS2), a heart rhythm disorder associated with sudden cardiac death. Mutations act either by reducing protein expression at the membrane and/or by perturbing the intricate gating properties of Kv11.1 channels. A number of clinical LQTS2-associated mutations have been reported in the first transmembrane segment (S1) of Kv11.1 channels, but the role of this region of the channel is largely unexplored. In part, this is due to problems defining the extent of the S1 helix, as a consequence of its low sequence homology with other Kv family members. Here, we used NMR spectroscopy and electrophysiological characterization to show that the S1 of Kv11.1 channels extends seven helical turns, from Pro-405 to Phe-431, and is flanked by unstructured loops. Functional analysis suggests that pre-S1 loop residues His-402 and Tyr-403 play an important role in regulating the kinetics and voltage dependence of channel activation and deactivation. Multiple residues within the S1 helix also play an important role in fine-tuning the voltage dependence of activation, regulating slow deactivation, and modulating C-type inactivation of Kv11.1 channels. Analyses of LQTS2-associated mutations in the pre-S1 loop or S1 helix of Kv11.1 channels demonstrate perturbations to both protein expression and most gating transitions. Thus, S1 region mutations would reduce both the action potential repolarizing current passed by Kv11.1 channels in cardiac myocytes, as well as the current passed in response to premature depolarizations that normally helps protect against the formation of ectopic beats.

Anisotropic diffusion in stretched hydrogels containing erythrocytes: evidence of cell-shape distortion recorded by PGSE NMR spectroscopy.

The remarkable flexibility of human red blood cells (RBCs) allows them to assume a range of shapes in normal and disease states. Biochemical mechanisms and energetic requirements associated with changes in RBC geometry are not well understood because of a lack of experimental procedures to fix and study cells in different morphological forms. By incorporating RBCs into stretchable gelatin hydrogels, we created conditions for adjustable elongation of their normal discocytic shape in all orientations. As the RBC-containing gels were stretched or compressed, the changes in the cell morphology were studied by using 1 H-PGSE-NMR spectroscopy. Measurements of the apparent diffusion coefficient of water along the three orthogonal directions revealed tuneable anisotropy in the environment of the hydrogel samples. Light microscopy was also used for recording the extent to which RBCs were distorted in a stretched gel that had been set around them. Having demonstrated the applicability of NMR diffusometry to detect morphological changes of immobilised cells, we have laid the groundwork for future investigations of controllably distorted RBCs. Specifically, we expect studies of metabolic and biophysical properties of the physically deformed cells, thus inferring the connection between intracellular physico-chemical processes and RBC morphology. Copyright © 2016 John Wiley & Sons, Ltd.

Na+ and solute diffusion in aqueous channels of Myverol bicontinuous cubic phase: PGSE NMR and computer modelling.

The apparent diffusion coefficients of 23 Na+ ions and the solute 2-fluoroethylamine present in the aqueous domain of a Myverol/water bulk bicontinuous cubic phase (BCP) were measured using pulsed field-gradient spin echo (PGSE) NMR spectroscopy. The measured values were dependent on the diffusion time interval, which is a characteristic of restricted diffusion. The translational motion of 23 Na+ and water in the aqueous channels of a cubic phase were simulated using a Monte-Carlo random walk algorithm, and the simulation results were compared with those from real PGSE NMR experiments. The simulations indicated that diffusion of 23 Na+ ions and water would appear to be restricted even on the shortest timescales available to PGSE NMR experiments. The micro-viscosity of the aqueous domain of the BCPs was estimated from the longitudinal relaxation times of 23 Na+ and 2-fluoroethylamine; this was three times higher than in free solution and suggests one of (but not the only) likely impediments to the release of hydrophilic drugs from stabilised aqueous dispersions of BCPs (cubosomes) when they are used therapeutically in vivo. Monte Carlo simulations of diffusive efflux from cubosomes suggest that the principal impediment to drug release is presented by a surfactant or lipid barrier at the cubosome surface, which separates the BCP aqueous channels from the bulk solution. The dynamics inferred from these studies informs quantitative predictions of drug delivery from cubosomes. Copyright © 2016 John Wiley & Sons, Ltd.