Directed Signaling Cascades in Monodisperse Artificial Eukaryotic Cells

The bottom-up assembly of multi-compartment artificial cells that are able to direct biochemical reactions along a specific spatial pathway remains a considerable engineering challenge. In this work, we address this with a microfluidic platform which is able to produce monodisperse multivesicular vesicles (MVVs) to serve as synthetic eukaryotic cells. Using a two-inlet polydimethylsiloxane (PDMS) channel design to co-encapsulate different populations of liposomes we are able to produce lipid-based MVVs in a high-throughput manner and with three separate inner compartments each containing a different enzyme: α-glucosidase, glucose oxidase, and horseradish peroxidase. We demonstrate the ability of these MVVs to carry out directed chemical communication between the compartments via the reconstitution of size-selective membrane pores. Therefore, the signal transduction, which is triggered externally, follows a specific spatial pathway between the compartments. We use this platform to study the effects of enzyme cascade compartmentalization by direct analytical comparison between bulk, one-, two-, and three-compartment systems. This microfluidic strategy to construct complex hierarchical structures is not only suitable to study compartmentation effects on biochemical reactions but is also applicable for developing advanced drug-delivery systems as well as minimal cells in the field of bottom-up synthetic biology.