Elucidation of cholesterol insertion efficiency into phase-transfer vesicles enables the rational design of phase-separated membranes as thermally-responsive platforms for artificial cell construction.

Giant unilamellar vesicles (GUVs) are a well-established tool for the study of membrane biophysics and are increasingly used as artificial cell models and functional units in biotechnology. This trend is driven by the development of emulsion-based generation methods such as Emulsion Phase Transfer (EPT), which facilitates the encapsulation of almost any water-soluble compounds (including biomolecules) regardless of size or charge, is compatible with droplet microfluidics, and allows GUVs with asymmetric bilayers to be assembled. However, the ability to control the composition of membranes formed via EPT remains an open question; this is key as composition gives rise to an array of biophysical phenomena which can be used to add functionality to membranes. Here, we evaluate the use of GUVs constructed via this method as a platform for phase behaviour studies and take advantage of composition-dependent features to engineer thermally-responsive GUVs. For the first time, we generate ternary GUVs (DOPC/DPPC/ cholesterol) using EPT, and by compensating for the lower cholesterol incorporation efficiencies, show that these possess the full range of phase behaviour displayed by electroformed GUVs. As a demonstration of the fine control afforded by this approach, we demonstrate release of dye and peptide cargo when ternary GUVs are heated through the immiscibility transition temperature, and show that release temperature can be tuned by changing vesicle composition. We show that GUVs can be individually addressed and release triggered using a laser beam. Our findings validate EPT as a suitable method for generating phase separated vesicles and provide a valuable proof-of-concept for engineering content release functionality into individually addressable vesicles, which could have a host of applications in the development of smart synthetic biosystems.

In this article we detail a robust high-throughput microfluidic platform capable of fabricating either symmetric or asymmetric giant unilamellar vesicles (GUVs) and characterise the mechanical properties of their membranes.

This talk will outline novel microfluidic strategies for biomembrane engineering that are capable of fabricating vesicles [1], droplet interface bilayer networks [2], multisomes [3] and artificial tissues [4] where parameters such as membrane asymmetry, membrane curvature, compartment connectivity and individual compartment contents can be controlled. Various bulk methods, such as extrusion, gentle hydration and electroformation, have been synonymous with the formation of lipid vesicles over recent years. However these strategies suffer from significant shortcomings associated with these processes including limited control of vesicle structural parameters such as size, lamellarity, membrane composition and internal contents. To address this technological bottleneck we have developed novel microfluidic platforms to form lipid vesicles in high-throughput with full control over the composition of both the inner and outer leaflet of the membrane thereby enabling the manufacture of symmetric and asymmetric vesicles. This is achieved by manufacturing microfluidic channels with a step junction, produced by double-layer photolithography, which facilitates the transfer of a W/O emulsion across an oil-water phase boundary and the self-assembly of a phospholipid bilayer. These platforms are being used to explore the role of asymmetry in biological systems [1] and study the engineering rules that regulate membrane mediated protein-protein interactions [5]. In addition, these technologies are enabling the construction of biological machines capable of acting as micro-reactors [6], environmental sensors and smart delivery vehicles [5] as well as complex multi-compartment artificial cells where the contents and connectivity of each compartment can be controlled. These compartments are separated by biological functional membranes that can facilitate transport between the compartments themselves and between the compartments and external environment. This approach has led to the development of multi-step enzymatic signalling cascades integrated into artificial cells leading to in-situ chemical synthesis [7]. The ability to compartmentalise synthetic cells is opening up the possibility of increasing our understanding of how this phenomena controls signal transduction and biological function in real cells by allowing us to manufacture protocells that more accurately capture the spatio-temporal structures of real cells. Finally we will present the development of droplet based artificial tissues that can be used to generate display materials based on biological components [4].

Giant unilamellar vesicles (GUVs) have a wide range of applications in biology and synthetic biology. As a result, new approaches for constructing GUVs using microfluidic techniques are emerging but there are still significant shortcomings in the control of fundamental vesicle structural parameters such as size, lamellarity, membrane composition and internal contents. We have developed a novel microfluidic platform to generate compositionally-controlled GUVs. Water-in-oil (W/O) droplets formed in a lipid-containing oil flow are transferred across an oil-water interface, facilitating the self-assembly of a phospholipid bilayer. In addition, for the first time we have studied the mechanical properties of the resultant lipid bilayers of the microfluidic GUVs. Using fluctuation analysis we were able to calculate the values for bending rigidity of giant vesicles assembled on chip and demonstrate that these correlate strongly with those of traditional low throughput strategies such as electroformation.