Microfluidic Formation of Artificial Cell Membranes and Compartments for Permeation Studies and Cascade Reactions

Abstract

Lipid membranes serve as dynamic boundaries between the extracellular environment and the cytosol, allowing cells to compartmentalize their biochemical functions. These lipid borders are effective barriers that maintain the composition and internal compartments of cells. Lipid membranes are selectively permeable to small molecules, enabling specific transport processes that control the selective passage of substances. In the field of bottom-up synthetic biology, lipid membranes are the scaffold to create minimal cells and to mimic reactions and processes at or across their membrane. Vesicles and droplet interface bilayers are generally used to study cells as simplified models, where microfluidic platforms can improve the creation and investigation of artificial cell membranes and their compartments. Using microfluidics, it is possible to control the experimental conditions more precisely than in bulk assays, and to generate artificial membranes that are very close to the thickness and composition of cellular lipid membranes. The integration of artificial cells in microfluidic systems is still challenging and existing methods have some shortcomings. The focus of this work was first to improve current platforms for the hydrodynamic trapping of vesicles created by swelling or electroformation. We used microfluidic devices to study the interaction of both peptides and toxins with lipid membranes, observing their permeation, membranolytic effects, and pore formation. These artificial cells, however, were largely polydisperse and the encapsulation of substances remained challenging. We therefore developed a method based on microfluidic droplet arrays to address this issue, where droplets were precisely placed with a spotting device in close proximity on the surface of a plate with micro fabricated cavities. Droplets were coated with a phospholipid monolayer and droplet interface bilayers formed when two or more droplets were brought into contact. These artificial cells were monodisperse, allowing straightforward encapsulation of substances, and enabling the tailoring of the membrane composition. We initially analyzed the artificial cell membranes and compartments trough an integrated fluorescence microscope. Subsequently, we developed a protocol to separate and extract the droplets, and to interface our platform with label free matrix assisted laser desorption/ionization and liquid chromatography mass spectrometry analysis. Translocation of molecules across membranes was tailored by the addition of the pore-forming toxin alpha-hemolysin to selected droplets. Our method delivered the automated formation of one- and two-dimensional multi compartmental droplet networks. We demonstrated the effectiveness of our approach by connecting droplets containing different compounds and enzyme solutions, and performing both translocation experiments and multistep enzymatic cascade reactions across the droplet network. Moreover, we investigated the permeation of molecules across the lipid membranes, an important component in drug development to predict the absorption of substances. Example model permeants were added to donor droplets, and the permeation across symmetric and asymmetric lipid bilayer membranes to acceptor droplets was monitored. With this approach, we were able to identify the permeability coefficients. Our platform has the potential to become a tool for the screening of drug membrane permeability in the future. The embedding of membrane proteins and the fusion of cell derived vesicles with the membrane are feasible research directions. Finally, the platform may prove useful for other studies such as the three dimensional assembling towards artificial cell colonies and creating complex artificial systems for bottom-up synthetic biology.