Liposome-assisted in-situ cargo delivery to artificial cells and cellular subcompartments

We report on liposome-mediated targeted delivery of membrane-impermeable constituents into surface-adhered giant lipid compartments, employed as artificial cells. Soluble cargo compounds are delivered by means of an open-space microfluidic device, which perfuses selected lipid compartments with loaded small unilamellar vesicles (SUVs) composed of cationic lipids. The SUV membranes fuse with the surface-adhered containers, merging their contents. We monitored the fusion process via Forster resonance energy transfer (FRET) by labeling both the membranes of the SUVs and the target compartments with a fluorophore pair. We established that, upon fusion, water-soluble dyes, fluorescently labeled genetic polymers, sugars and proteins carried by the SUVs can be successfully internalized at high yield. Finally, by transferring carbonic anhydrase (CA) to the giant lipid compartments, enzymatic hydrolysis of the prefluorescent carboxyfluorescein diacetate (CFDA) is demonstrated by the emission intensity increase emanating from the product carboxyfluorescein (CF). Spontaneous subcompartmentalization occurred during liposomal delivery of the enzyme, leading to CF formation in an organelle-like subcompartment. The reported targeted delivery technique enables chemical reactions and cell-free gene expression in synthetic cell models with unprecedented ease and precision, and opens pathways to protocell architectures with distinct functional subcompartments in the context of origins of life research.