Alexandru Zabara

Controlling molecular transport and sustained drug release in lipid-based liquid crystalline mesophases

Lipid-based lyotropic liquid crystals, also referred to as reversed liquid crystalline mesophases, such as bicontinuous cubic, hexagonal or micellar cubic phases, have attracted deep interest in the last few decades due to the possibility of observing these systems at thermodynamic equilibrium in excess water conditions. This becomes of immediate significance for applications in the colloidal environment, such as in the food, cosmetic and pharmaceutical arenas. One possible application regarded as very promising is that of controlled delivery of functional ingredients. Different crystallographic structures of the lipid mesophase give access to different diffusion coefficients and distinct diffusion modes. It becomes thus crucial to engineer the space group of the mesophase in a controlled way, and ideally, in a stimuli-responsive manner. In this article we review the state of the art on diffusion and molecular transport in lipid-based mesophases and we discuss recent contributions to the controlled delivery of molecules and colloids through these systems. In particular we focus on the different available strategies relying on either endogenous or exogenous stimuli to induce changes in the symmetry and transport properties of lipid-based mesophases and we discuss the impact and implications this may have on controlled drug delivery.

Perforated bicontinuous cubic phases with pH-responsive topological channel interconnectivity.

Lipidic lyotropic liquid crystals are at the frontline of current research for release of target therapeutic molecules due to their unique structural complexity and the possibility of engineering stimuli-triggered release of both hydrophilic and hydrophobic molecules. One of the most suitable lipidic mesophases for the encapsulation and delivery of drugs is the reversed double diamond bicontinuous cubic phase, in which two distinct and parallel networks of ∼4 nm water channels percolate independently through the lipid bilayers, following a Pn3m space group symmetry. In the unperturbed Pn3m structure, the two sets of channels act as autonomous and non-communicating 3D transport pathways. Here, a novel type of bicontinuous cubic phase is introduced, where the presence of OmpF membrane proteins at the bilayers provides unique topological interconnectivities among the two distinct sets of water channels, enabling molecular active gating among them. By a combination of small-angle X-ray scattering, release and ion conductivity experiments, it is shown that, without altering the Pn3m space group symmetry or the water channel diameter, the newly designed perforated bicontinuous cubic phase attains transport properties well beyond those of the standard mesophase, allowing faster, sustained release of bioactive target molecules. By further exploiting the pH-mediated pore-closing response mechanism of the double amino acid half-ring architecture in the membrane protein, the pores of the perforated mesophase can be opened and closed with a pH trigger, enabling a fine modulation of the transport properties by only moderate changes in pH, which could open unexplored opportunities in the targeted delivery of bioactive compounds.

Plenty of room to crystallize: swollen lipidic mesophases for improved and controlled in-meso protein crystallization

The lipidic cubic phase method for protein crystallization, also known as “in-meso”, has become widely recognized over the last decade, with clear advantages over other crystallization methods, especially when applied to membrane proteins. However, its advances are severely thwarted by the major limitation of the small size of the mesophase nanofluidic aqueous channels, preventing crystallization of large hydrophilic proteins and reconstitution of membrane proteins with large extramembrane domains. Here we show that modulation and increase of the size of the ordered mesophase hydrophilic domains, in a well-controlled way, by doping the hosting lipids with hydration-enhancing surfactants can open up the in-meso crystallization to a wide array of previously inaccessible proteins. To demonstrate unambiguously the potential of this strategy, we design as a hosting mesophase a reverse bicontinuous cubic phase of double diamond symmetry (Pn3m) with a well-defined and controllable channel diameter, by mixing monoglycerides and octyl glucoside surfactants, and show for the first time how this system can serve for the in-meso crystallization of the progressively larger β-lactoglobulin, thaumatin and elastase protein series.

Tuning in-meso-Crystallized Lysozyme Polymorphism by Lyotropic Liquid Crystal Symmetry

Lipid-based lyotropic liquid crystals (LLCs) show great potential for applications in fields as diverse as food technology, cosmetics, pharmaceutics, or structural biology. Recently, these systems have provided a viable alternative to the difficult process of membrane protein crystallization, owing to their similarities with cell membranes. Nonetheless, the process of in-meso crystallization of proteins still remains poorly understood. In this study, we demonstrate that in-meso crystal morphologies of lysozyme (LSZ), a model hydrophilic protein, can be controlled by both the composition and symmetry of the mesophase, inferring a possible general influence of the LLC space group on the protein crystal polymorphism. Lysozyme was crystallized in-meso from three common LLC phases (lamellar, inverse hexagonal, and inverse bicontinuous cubic) composed of monolinolein and water. Different mixing ratios of mesophase to crystallization buffer were used in order to tune crystallization both in the bulk mesophase and in excess water conditions. Two distinct mechanisms of crystallization were shown to take place depending on available water in the mesophases. In the bulk mesophases, protein nuclei form and grow within structural defects of the mesophase and partially dehydrate the system inducing order-to-order transitions of the liquid crystalline phase toward stable symmetries in conditions of lower hydration. The formed protein crystals eventually macrophase separate from the mesophase allowing the system to reach its final symmetry. On the other hand, when excess water is available, protein molecules diffuse from the water channels into the excess water, where the crystallization process can take place freely, and with little to no effect on the structure and symmetry of the lyotropic liquid crystals.

Modulating the crystal size and morphology of in meso-crystallized lysozyme by precisely controlling the water channel size of the hosting mesophase

We explore the effects of increased protein mobility inside the aqueous channels of ordered lyotropic liquid crystals (LLC) of cubic Pn3m symmetry on the process of in meso crystallization of lysozyme proteins. The protein confinement within the channels is released by swelling the aqueous domains by doping the mesophase with a hydration-modulating agent that causes a near twofold increase in the diameter of the water channels. By means of small angle X-ray scattering and cross-polarized optical microscopy, we then show that increased diffusion of both protein and water molecules within the bulk hosting LLC leads to the formation of not only larger protein crystals but also crystals belonging to different polymorphic forms.

Phase Behavior of a Designed Cyclopropyl Analogue of Monoolein: Implications for Low-Temperature Membrane Protein Crystallization†

Lipidic cubic phases (LCPs) are used in areas ranging from membrane biology to biodevices. Because some membrane proteins are notoriously unstable at room temperature, and available LCPs undergo transformation to lamellar phases at low temperatures, development of stable low-temperature LCPs for biophysical studies of membrane proteins is called for. Monodihydrosterculin (MDS) is a designer lipid based on monoolein (MO) with a configurationally restricted cyclopropyl ring replacing the olefin. Small-angle X-ray scattering (SAXS) analyses revealed a phase diagram for MDS lacking the high-temperature, highly curved reverse hexagonal phase typical for MO, and extending the cubic phase boundary to lower temperature, thereby establishing the relationship between lipid molecular structure and mesophase behavior. The use of MDS as a new material for LCP-based membrane protein crystallization at low temperature was demonstrated by crystallizing bacteriorhodopsin at 20 °C as well as 4 °C.

Modulating the structural properties of β-d-glucan degradation products by alternative reaction pathways

The aim of the present study was to compare the degradation of β-D-glucan induced by hydroxyl radical to the degradation induced by heat treatment. β-D-Glucan was quickly and widely degraded by the action of hydroxyl radicals produced by a Fenton system at 85 °C, while thermal hydrolysis at 85 °C induced slow β-D-glucan depolymerization. The hydroxyl radical-induced degradation of β-D-glucan was accompanied by the formation of peroxyl radicals and new oxidized functional groups (i.e. lactones, carboxylic acids, ketones and aldehydes), as detected by ESR and NMR, respectively. In contrast, no changes in the monomer chemical structure of β-D-glucan were observed upon thermal hydrolysis. Therefore, different mechanisms are proposed for the oxidative cleavage of β-D-glucan, which are initiated by the presence of an unpaired electron on the anomeric carbon.

Reconstitution of OmpF membrane protein on bended lipid bilayers: perforated hexagonal mesophases

Membrane proteins have been reconstituted on lipid bilayers with zero mean-curvature (cubic phases or vesicles). Here we show that reconstitution of pore-forming membrane proteins can also occur on highly curved lipidic bilayers of reverse hexagonal mesophases, for which the mean-curvature is significantly different from zero. We further show that the membrane protein provides unique topological interconnectivities between the aqueous nanochannels, significantly enhancing mesophase transport properties.

Effect of Phytosterols on the Crystallization Behavior of Oil-in-Water Milk Fat Emulsions

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.

Active Gating, Molecular Pumping, and Turnover Determination in Biomimetic Lipidic Cubic Mesophases with Reconstituted Membrane Proteins

Understanding the mechanisms controlling molecular transport in bioinspired materials is a central topic in many branches of nanotechnology. In this work, we show that biomolecules of fundamental importance in biological processes, such as glucose, can be transported in an active, controlled, and selective manner across macroscopic lipidic cubic mesophases, by correctly reconstituting within them their corresponding membrane protein transporters, such as Staphylococcus epidermidis (GlcPSe). Importantly, by duly exploiting the symporter properties of GlcPSe of coupled glucose/H+ transport, the diffusion of glucose can further be tuned by independent physiological stimuli, such as parallel or antiparallel pH gradients, offering an important model to study molecular exchange processes in cellular machinery. We finally show that by measuring the transport properties of the lipidic mesophases with and without the GlcPSe membrane protein reconstituted within, it becomes possible to determine its intrinsic conductance. We generalize these findings to other membrane proteins from the antiporters family, such as the bacterial ClC exchanger from Escherichia coli (EcClC), providing a robust method for evaluating the turnover rate of the membrane proteins in general.