Membraneless organelles formed by liquid-liquid phase separation are dynamic structures that are employed by cells to spatiotemporally regulate their interior. Indeed, complex coacervation-based phase separation is involved in a multitude of biological tasks ranging from photosynthesis to cell division to chromatin organization, and more. Here, we use an on-chip microfluidic method to control and study the formation of membraneless organelles within liposomes, using pH as the main control parameter. We show that a transmembrane proton flux that is created by a stepwise change in the external pH can readily bring about the coacervation of encapsulated components in a controlled manner. We employ this strategy to induce and study electrostatic as well as hydrophobic interactions between the coacervate and the lipid membrane. Electrostatic interactions using charged lipids efficiently recruit coacervates to the membrane and restrict their movement along the inner leaflet. Hydrophobic interactions via cholesterol-tagged RNA molecules provide even stronger interactions, causing coacervates to wet the membrane and affect the local lipid-membrane structure, reminiscent of coacervate-membrane interactions in cells. The presented technique of pH-triggered coacervation within cell-sized liposomes may find applications in synthetic cells and in studying biologically relevant phase separation reactions in a bottom-up manner.

Liquid-liquid phase separation (LLPS), especially coacervation, plays a crucial role in cell biology, as it forms numerous membraneless organelles in cells. Coacervates play an indispensable role in regulating intracellular biochemistry, and their dysfunction is associated with several diseases. Understanding of the LLPS dynamics would greatly benefit from controlled in vitro assays that mimic cells. Here, we use a microfluidics-based methodology to form coacervates inside cell-sized (~10 µm) liposomes, allowing control over the dynamics. Protein-pore-mediated permeation of small molecules into liposomes triggers LLPS passively or via active mechanisms like enzymatic polymerization of nucleic acids. We demonstrate sequestration of proteins (FtsZ) and supramolecular assemblies (lipid vesicles), as well as the possibility to host metabolic reactions (β-galactosidase activity) inside coacervates. This coacervate-in-liposome platform provides a versatile tool to understand intracellular phase behavior, and these hybrid systems will allow engineering complex pathways to reconstitute cellular functions and facilitate bottom-up creation of synthetic cells.