Intravesicular Solute Delivery and Surface Area Regulation in Giant Unilamellar Vesicles Driven by Cycles of Osmotic Stresses.

Phospholipid bilayers are dynamic cellular components that undergo constant changes in their topology, facilitating a broad diversity of physiological functions including endo- and exocytosis, cell division, and intracellular trafficking. These shape transformations consume energy, supplied invariably by the activity of proteins. Here, we show that cycles of oppositely directed osmotic stresses─unassisted by any protein activity─can induce well-defined remodeling of giant unilamellar vesicles, minimally recapitulating the phenomenologies of surface area homeostasis and macropinocytosis. We find that a stress cycle consisting of deflationary hypertonic stress followed by an inflationary hypotonic one prompts an elaborate sequence of membrane shape changes ultimately transporting molecular cargo from the outside into the intravesicular milieu. The initial osmotic deflation produces microscopic spherical invaginations. During the subsequent inflation, the first subpopulation contributes area to the swelling membrane, thereby providing a means for surface area regulation and tensional homeostasis. The second subpopulation vesiculates into the lumens of the mother vesicles, producing pinocytic vesicles. Remarkably, the gradients of solute concentrations between the GUV and the daughter pinocytic vesicles create cascades of water current, inducing pulsatory transient poration that enable solute exchange between the buds and the GUV interior. This results in an efficient water-flux-mediated delivery of molecular cargo across the membrane boundary. Our findings suggest a primitive physical mechanism for communication and transport across protocellular compartments driven only by osmotic stresses. They also suggest plausible physical routes for intravesicular, and possibly intracellular, delivery of ions, solutes, and molecular cargo stimulated simply by cycles of osmotic currents of water.

Production of Lipid Constructs by Design via Three-Dimensional Nanoprinting.

Atomic force microscopy (AFM) in conjunction with microfluidic delivery was utilized to produce three-dimensional (3D) lipid structures following a custom design. While AFM is well-known for its spatial precision in imaging and 2D nanolithography, the development of AFM-based nanotechnology into 3D nanoprinting requires overcoming the technical challenges of controlling material delivery and interlayer registry. This work demonstrates the concept of 3D nanoprinting of amphiphilic molecules such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Various formulations of POPC solutions were tested to achieve point, line, and layer-by-layer material delivery. The produced structures include nanometer-thick disks, long linear spherical caps, stacking grids, and organizational chiral architectures. The POPC molecules formed stacking bilayers in these constructions, as revealed by high-resolution structural characterizations. The 3D printing reached nanometer spatial precision over a range of 0.5 mm. The outcomes reveal the promising potential of our designed technology and methodology in the production of 3D structures from nanometer to continuum, opening opportunities in biomaterial sciences and engineering, such as in the production of 3D nanodevices, chiral nanosensors, and scaffolds for tissue engineering and regeneration.

Impact of Surface Polarity on Lipid Assembly under Spatial Confinement.

Molecular dynamics (MD) simulations in the MARTINI model are used to study the assembly of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) molecules under spatial confinement, such as during solvent evaporation from ultrasmall (femtoliter quantity) droplets. The impact of surface polarity on molecular assembly is discussed in detail. To the best of our knowledge, this work represents the first of its kind. Our results reveal that solvent evaporation gives rise to the formation of well-defined stacks of lipid bilayers in a smectic alignment. These smectic mesophases form on both polar and nonpolar surfaces but with a notable distinction. On polar surfaces, the director of the stack is oriented perpendicular to the support surface. By contrast, the stacks orient at an angle on the nonpolar surfaces. The packing of head groups on surfaces and lipid molecular mobility exhibits significant differences as surface polarity changes. The role of glycerol in the assembly and stability is also revealed. The insights revealed from the simulation have a significant impact on additive manufacturing, biomaterials, model membranes, and engineering protocells. For example, POPC assemblies via evaporation of ultrasmall droplets were produced and characterized. The trends compare well with the bilayer stack models. The surface polarity influences the local morphology and structures at the interfaces, which could be rationalized via the molecule-surface interactions observed from simulations.