Super-swelled lyotropic single crystals

Abstract

Lipids self-assemble into diverse supramolecular structures that exhibit thermotropic and/or lyotropic behavior. Lyotropic mesophases, where membranes conform to periodic minimal surfaces dividing two nonpenetrating aqueous subspaces, are arguably one of the most intriguing phases of lipid materials. Traditional 3D bicontinuous cubic lipid materials appear as a polycrystal of varying degrees of order. When exposed to water, the properties of the molecular building blocks of the membrane determine specific swelling limits setting the lattice dimensions at about 15 nm. This limited swelling severely impairs their application as delivery vehicles of large drugs or as matrices for guiding protein crystallization. We report the discovery of self-assembly strategies leading to the emergence of lipid bicontinuous single crystals with unprecedented swelling capacity. The conventional strategy to increase unit cell size is tweaking membrane composition to include charged building blocks, a process to achieve electrostatic-driven swelling. In this paper, we demonstrate that controlling self-assembly external conditions when coupled to membrane composition yields 3D bicontinuous cubic phases that swell up to lattice dimensions of 68 nm. Importantly, and contrary to what is perceived for soft lyotropic materials in general, the self-assembly methodology enables the development of large super-swelled monocrystals. Utilizing small-angle X-ray scattering and cryoelectron microscopy, we underpin three crucial factors dictating the stabilization of super-swelled lipid bicontinuous cubic single crystals: () organic solvent drying speed, () membrane charge density, and () polyethylene glycol-conjugated lipids amount.

Microfluidics Synthesis of Gene Silencing Cubosomes

“…This ternary mixture emerged as a fruitful polymorphic system, and all three symmetry groups ( Ia3d, Pn3m, and Im3m ) of bicontinuous cubic phases could be stabilized in excess water. 14,39 This is in contrast to pure GMO, where only the Pn3m is stable. 40 Importantly, the formation of the cubosomes was successful simply by ultrachilled sonication, a method completely analogous to producing a liposome.…”

The stabilization of primitive bicontinuous cubic phases with tunable swelling over a wide composition range

“…also showed that larger water channels with lower curvature are more favorable for inclusion of guest molecules that are rather stiff such as gold nanoparticles. 25 This work has direct relevance for designing both bulk and lipid nanoparticle delivery systems and contributes to the understanding of swollen lipid bicontinuous cubic phases.…”

“…30 Recently we have shown that a lipid mixture of GMO, DOTAP (1,2-dioleoyl-3-trimethylammonium propane), and GMO-PEG (custom designed GMO covalently linked to polyethylene glycol 2 kDa) in excess water enables the stabilization of highly ordered bicontinuous cubic phases in excess water of all three symmetry groups (gyroid, diamond, and primitive), with tunable lattice dimensions ( a Q = 150–680 Å). 6,25 The resultant cubic phase retains order, as in a single-crystal, and lattice dimension for an extended period of time (> 6 months). Tuning of both symmetry group and lattice size can be achieved by varying composition as well as processing conditions.…”

“…In this paper, we employ small angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (Cryo-EM) to explore the ternary lipid mixture of GMO, DOTAP, and negatively charged 1,2-dioleoyl- sn-giycero -3-phosphoethanolamine- N -[methoxy(polyethylene glycol)-2000 kDa] (DOPE-PEG) in excess water in a compositional regime relevant for the design of siRNA 6,8 and drug 35 delivery systems. All three lipids are commercially available, in contrast to the analogous ternary lipid mixture used in our previous studies 6,8,25 where custom synthetized neutral GMO-PEG was used as the PEGylated lipid. Fig.…”

Design of ultra-swollen lipidic mesophases for the crystallization of membrane proteins with large extracellular domains

“…However, the largest swelling was observed at high temperature (i.e., 35–45 °C 23 and 55 °C 24 ) and the only symmetry observed was the primitive Im3m cubic phase, both factors being unsuitable for in meso membrane protein crystallization, which typically requires lower temperatures and Pn3m symmetry. By combining the electrostatic swelling effects with epitaxial growth from capillary walls, very recently Kim et al 27 reported super swollen mesophases of Im3m, Ia3d, and Pn3m symmetries with lattice parameters up to of 68.4 nm, and the unusual coexistence of double-gyroid symmetries with excess water 27 . Nonetheless, these conditions were reported far from equilibrium, and the meta-stability of the system reduces its applicability for membrane protein crystallization, which may require weeks for crystals to nucleate and growth: indeed authors did not consider it for this specific use and to date, in meso crystallization of membrane proteins from thermodynamically stable ultra-swollen LCP is still to be achieved.…”