Osmotic Pressure Enables High-Yield Assembly of Giant Vesicles in Solutions of Physiological Ionic Strengths.

Giant unilamellar vesicles (GUVs) are micrometer-scale minimal cellular mimics that are useful for bottom-up synthetic biology and drug delivery. Unlike assembly in low-salt solutions, assembly of GUVs in solutions with ionic concentrations of 100-150 mM Na/KCl (salty solutions) is challenging. Chemical compounds deposited on the substrate or incorporated into the lipid mixture could assist in the assembly of GUVs. Here, we investigate quantitatively the effects of temperature and chemical identity of six polymeric compounds and one small molecule compound on the molar yields of GUVs composed of three different lipid mixtures using high-resolution confocal microscopy and large data set image analysis. All the polymers moderately increased the yields of GUVs either at 22 or 37 °C, whereas the small molecule compound was ineffective. Low-gelling temperature agarose is the singular compound that consistently produces yields of GUVs of greater than 10%. We propose a free energy model of budding to explain the effects of polymers in assisting the assembly of GUVs. The osmotic pressure exerted on the membranes by the dissolved polymer balances the increased adhesion between the membranes, thus reducing the free energy for bud formation. Data obtained by modulating the ionic strength and ion valency of the solution shows that the evolution of the yield of GUVs supports our model's prediction. In addition, polymer-specific interactions with the substrate and the lipid mixture affects yields. The uncovered mechanistic insights provide a quantitative experimental and theoretical framework to guide future studies. Additionally, this work shows a facile means for obtaining GUVs in solutions of physiological ionic strengths.

Cellulose Abetted Assembly and Temporally Decoupled Loading of Cargo into Vesicles Synthesized from Functionally Diverse Lamellar Phase Forming Amphiphiles.

Self-assembled micrometer-scale vesicles composed of lamellar phase forming amphiphiles are useful as chemical microreactors, as minimal artificial cells, as protocell mimics for studies of the origins of life, and as vehicles for the targeted delivery of drugs. Given their varied uses, discovery of a universal mechanism that is simple, rapid, and that produces vesicles from a large variety of amphiphiles with different chemical and physical properties at high yield is extremely desirable. Here we show that cellulose, in the form of cellulose paper, facilitates the assembly of membranous vesicles 5-20 µm in diameter from scientifically and technologically important amphiphiles of diverse chemical structures and functionality such as fatty acids (fatty acid vesicles), amphiphilic diblock copolymers, and amphiphilic triblock copolymers (polymersomes). Assembly of vesicles occurred within 90 min of placing the amphiphile-coated cellulose paper into aqueous solutions. Varying thermal and chemical conditions, however, are required for the high-yield assembly of vesicles from the different amphiphiles. The vesicles, when attached to cellulose fibers, have membranes that remain unsealed. This topological characteristic of the vesicles grown on paper allowed the scalable separation of the process of growth from the process of loading cargo (temporally decoupled growth and loading). We demonstrate a temporally decoupled process to rapidly produce large quantities of protein-loaded polymersomes on the benchtop by using high temperatures to accelerate the growth of the polymersomes and subsequently milder temperatures during diffusive loading of the protein cargo.

Shape Transformations of Lipid Bilayers Following Rapid Cholesterol Uptake.

High cholesterol levels in the blood increase the risk of atherosclerosis. A common explanation is that the cholesterol increase in the plasma membrane perturbs the shape and functions of cells by disrupting the cell signaling pathways and the formation of membrane rafts. In this work, we show that after enhanced transient uptake of cholesterol, mono-component lipid bilayers change their shape similarly to cell membranes in vivo. The bilayers either expel lipid protrusions or spread laterally as a result of the ensuing changes in their lipid density, the mechanical constraints imposed on them, and the properties of cyclodextrin used as a cholesterol donor. In light of the increasingly recognized link between membrane tension and cell behavior, we propose that the physical adaptation of the plasma membrane to cholesterol uptake may play a substantial role in the biological response.

Glycans pattern the phase behaviour of lipid membranes.

Hydrated networks of glycans (polysaccharides)--in the form of cell walls, periplasms or gel-like matrices--are ubiquitously present adjacent to cellular plasma membranes. Yet, despite their abundance, the function of glycans in the extracellular milieu is largely unknown. Here we show that the spatial configuration of glycans controls the phase behaviour of multiphase model lipid membranes: inhomogeneous glycan networks stabilize large lipid domains at the characteristic length scale of the network, whereas homogeneous networks suppress macroscopic lipid phase separation. We also find that glycan-patterned phase separation is thermally reversible--thus indicating that the effect is thermodynamic rather than kinetic--and that phase patterning probably results from a preferential interaction of glycans with ordered lipid phases. These findings have implications for membrane-mediated transport processes, potentially rationalize long-standing observations that differentiate the behaviour of native and model membranes and may indicate an intimate coupling between cellular lipidomes and glycomes.

Lipid Bilayers Are Long-Lived on Solvent Cleaned Plasma-Oxidized poly(dimethyl)siloxane (ox-PDMS).

Although it is well known that phospholipids self-assemble on hydrophilic plasma-oxidized PMDS surfaces (ox-PDMS) to form cell membrane mimetic bilayers, the temporal stability of phospholipid membranes on these surfaces is unknown. Here we report that phospholipid bilayers remain stable on solvent-cleaned ox-PDMS for at least 132 hours after preparation. Absent solvent cleaning, the bilayers were stable for only 36 hours. We characterized the phospholipid bilayers, i) through quantitative comparative analysis of the fluorescence intensity of phospholipid bilayers on ox-PDMS and phospholipid monolayers on native PDMS and, ii) through measurements of the diffusive mobility of the lipids through fluorescence recovery after photobleaching (FRAP). The fluorescence intensity of the phospholipid layer remained consistent with that of a bilayer for 132 hours. The evolution of the diffusive mobility of the phospholipids in the bilayer on ox-PDMS over time was similar to lipids in control bilayers prepared on glass surfaces. Solvent cleaning was essential for the long-term stability of the bilayers on ox-PDMS. Without cleaning in acetone and isopropanol, phospholipid bilayers prepared on ox-PDMS surfaces peeled off in large patches within 36 hours. Importantly, we find that phospholipid bilayers supported on solvent-cleaned ox-PDMS were indistinguishable from phospholipid bilayers supported on glass for at least 36 hours after preparation. Our results provide a link between the two common surfaces used to prepare in vitro biomimetic phospholipid membranes-i) glass surfaces used predominantly in fundamental biophysical experiments, for which there is abundant physicochemical information, with ii) ox-PDMS, the dominant material used in practical, applications-oriented systems to build micro-devices, topographically-patterned surfaces, and biosensors where there is a dearth of information.

Novel Application of Cellulose Paper As a Platform for the Macromolecular Self-Assembly of Biomimetic Giant Liposomes.

We report a facile and scalable method to fabricate biomimetic giant liposomes by using a cellulose paper-based materials platform. Termed PAPYRUS for Paper-Abetted liPid hYdRation in aqUeous Solutions, the method is general and can produce liposomes in various aqueous media and at elevated temperatures. Encapsulation of macromolecules and production of liposomes with membranes of complex compositions is straightforward. The ease of manipulation of paper makes practical massive parallelization and scale-up of the fabrication of giant liposomes, demonstrating for the first time the surprising usefulness of paper as a platform for macromolecular self-assembly.

Using magnetic levitation for non-destructive quality control of plastic parts.

Magnetic levitation (MagLev) enables rapid and non-destructive quality control of plastic parts. The feasibility of MagLev as a method to: i) rapidly assess injection-molded plastic parts for defects during process optimization, ii) monitor the degradation of plastics after exposure to harsh environmental conditions, and iii) detect counterfeit polymers by density is demonstrated.

Polymer-based mesh as supports for multi-layered 3D cell culture and assays.

Three-dimensional (3D) culture systems can mimic certain aspects of the cellular microenvironment found in vivo, but generation, analysis and imaging of current model systems for 3D cellular constructs and tissues remain challenging. This work demonstrates a 3D culture system-Cells-in-Gels-in-Mesh (CiGiM)-that uses stacked sheets of polymer-based mesh to support cells embedded in gels to form tissue-like constructs; the stacked sheets can be disassembled by peeling the sheets apart to analyze cultured cells-layer-by-layer-within the construct. The mesh sheets leave openings large enough for light to pass through with minimal scattering, and thus allowing multiple options for analysis-(i) using straightforward analysis by optical light microscopy, (ii) by high-resolution analysis with fluorescence microscopy, or (iii) with a fluorescence gel scanner. The sheets can be patterned into separate zones with paraffin film-based decals, in order to conduct multiple experiments in parallel; the paraffin-based decal films also block lateral diffusion of oxygen effectively. CiGiM simplifies the generation and analysis of 3D culture without compromising throughput, and quality of the data collected: it is especially useful in experiments that require control of oxygen levels, and isolation of adjacent wells in a multi-zone format.

The effect of double-chain surfactants on armored bubbles: a surfactant-controlled route to colloidosomes.

We find that the gas phases of air bubbles covered with anionic or cationic polystyrene latex particles dissolve on exposure to cationic and catanionic surfactants. The particles on the bubble interface are released as singlets or aggregates when the surfactant has a single hydrophobic chain, while porous colloidal capsules (colloidosomes) with the same aqueous phase inside as out are obtained when the surfactant has two hydrophobic chains. The formation of colloidosomes from the particle-covered bubbles does not appear to depend significantly on the charge of the particles, which makes it unlikely that bilayers of surfactant are stabilizing the colloidosome. While the exact mechanism of formation remains an open question, our method is a simple one-step process for obtaining colloidosomes from particle-covered bubbles.

Controlled assembly of jammed colloidal shells on fluid droplets.

Assembly of colloidal particles on fluid interfaces is a promising technique for synthesizing two-dimensional microcrystalline materials useful in fields as diverse as biomedicine, materials science, mineral flotation and food processing. Current approaches rely on bulk emulsification methods, require further chemical and thermal treatments, and are restrictive with respect to the materials used. The development of methods that exploit the great potential of interfacial assembly for producing tailored materials have been hampered by the lack of understanding of the assembly process. Here we report a microfluidic method that allows direct visualization and understanding of the dynamics of colloidal crystal growth on curved interfaces. The crystals are periodically ejected to form stable jammed shells, which we refer to as colloidal armour. We propose that the energetic barriers to interfacial crystal growth and organization can be overcome by targeted delivery of colloidal particles through hydrodynamic flows. Our method allows an unprecedented degree of control over armour composition, size and stability.