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Vesicle-stabilized all-aqueous emulsion droplets are appealing as bioreactors because they provide uniform encapsulation via equilibrium partitioning without restricting diffusion in and out of the interior. These properties rely on the composition of the aqueous two-phase system (ATPS) chosen for the emulsion and the structure of the interfacial liposome layer, respectively. Here, we explore how changing the aqueous two-phase system from a standard poly(ethyleneglycol), PEG, 8 kDa/dextran 10 kDa ATPS to PEG 8 kDa/Ficoll 70 kDa or PEG 8 kDa/Na2SO4 systems impacts droplet uniformity and partitioning of a model solute (U15 oligoRNA). We also compare liposomes formed by two different methods, both of which begin with multilamellar, polydisperse vesicles formed by gentle hydration: (1) extrusion, which produced vesicles of 150 nm average diameter, and (2) vortexing, which produced vesicles of 270 nm average diameter. Our data illustrate that while droplet uniformity and stability are somewhat better for samples based on extruded vesicles, extrusion is not necessary to create functional microreactors, as emulsions stabilized with vortexed liposomes are just as effective at solute partitioning and allow diffusion across the droplet's liposome corona. This work expands the compositions possible for liposome-stabilized, all-aqueous emulsion droplet bioreactors, making them amenable to a wider range of potential reactions. Replacing the liposome extrusion step with vortexing can reduce time and cost of bioreactor production with only modest reductions in emulsion quality.

Living organisms direct the location, structure, and properties of biominerals by tightly controlling reactant concentration profiles, biopolymer identity and availability, and other aspects of rea...

An astounding variety of cellular contexts converge to the process of liquid-liquid phase separation for the creation of new functional levels of organization. But the kinetic pathways by which intracellular phase separation proceeds – typically in physically confined and macromolecularly crowded volumes of topologically closed cellular and intracellular compartments –remain incompletely understood. Here, we monitor the dynamics of liquid-liquid phase separation of mixtures of phase-separating polymers (i.e., polyethyleneglycol and dextran) inside all-synthetic, cell-sized giant unilamellar vesicles in real-time. We dynamically trigger phase separation by subjecting an initially homogeneous polymer solution inside vesicles to an abrupt osmotic quench. The latter removes water and elevates polymer concentrations in the phase-coexistence regime thereby initiating a segregative phase separation of the polymers. We find that the ensuing relaxation – en route to the new equilibrium – is non-trivially modulated by a dynamic interplay between the coarsening of the evolving droplet phase and the interactive membrane boundary. The early trajectory of droplet coarsening exhibit significant acceleration, but a competing process of membrane-droplet interactions – one in which the membrane boundary is preferentially wetted by one of the incipient phases – dynamically arrests the progression and deforms the membrane. As a result, a novel multi-bud morphology, reminiscent of cellular blebs, decorate the vesicle surface. Furthermore, when the vesicles are composed of phase-separating mixtures of common lipids, the three-dimensional liquid-liquid phase separation within the vesicular interior becomes coupled to the membrane’s compositional degrees of freedom producing microphase-separated membrane textures. This coupling of bulk and surface phase separation processes suggests a new physical principle by which liquid-liquid phase separation inside living cells might be dynamically regulated and materially communicated inside-out to the cellular boundaries.

Mineral deposition within living cells relies on control over the distribution and availability of precursors as well as the location and rates of nucleation and growth. This control is provided in large part by biomolecular chelators, which bind precursors and regulate their availability, and compartmentalization within specialized mineralizing vesicles. Biomimetic mineralization in self-assembled lipid vesicles is an attractive means of studying the mineralization process, but has proven challenging due to vesicle heterogeneity in lamellarity, contents, and size across a population, difficulties encapsulating high and uniform precursor concentrations, and the need to transport reagents across an intact lipid bilayer membrane. Here, we report the use of liposome-stabilized all-aqueous emulsion droplets as simple artificial mineralizing vesicles (AMVs). These biomimetic microreactors allow the entry of precursors while retaining a protein catalyst by equilibrium partitioning between internal and external polymer-rich phases. Small molecule chelators with intermediate binding affinity were employed to control Ca(2+) availability during CaCO3 mineralization, providing protection against liposome aggregation while allowing CaCO3 formation. Mineral deposition was limited to the AMV interior, due to localized production of CO3(2-) by compartmentalized urease. Particle formation was uniform across the entire population of AMVs, with multiple submicrometer amorphous CaCO3 particles produced in each one. The all-aqueous emulsion-based approach to biomimetic giant mineral deposition vesicles introduced here should be adaptable for enzyme-catalyzed synthesis of a wide variety of materials, by varying the metal ion, enzyme, and/or chelator.