Membrane composition and osmotic conditions jointly regulate encapsulation and translation efficiency in GUV based...

Giant unilamellar vesicles (GUVs) are widely used as cell-sized compartments for artificial cell models that encapsulate transcription and translation reactions. Although isotonic sugar conditions between inner and outer aqueous phases are commonly adopted in the water-in-oil emulsion transfer method, their functional significance has not been systematically examined. Here, we quantitatively analyzed how inner and outer sugar concentrations and membrane composition affect GUV formation, molecular encapsulation, compartment stability, and translation activity using flow cytometry. An inner sucrose-to-outer glucose ratio of 1:2 enhanced GUV formation efficiency and molecular retention compared with isotonic conditions. In contrast, encapsulation efficiency within formed GUVs was primarily determined by the inner sugar concentration and was higher under low-sucrose conditions. GFP translation analysis revealed that translation output depends mainly on encapsulation efficiency and the fraction of translation-competent compartments, rather than molecular leakage during incubation. Incorporation of the negatively charged lipid POPG further improved GUV formation and translation efficiency. These results demonstrate that functional GUV-based artificial cell systems require integrated optimization of membrane composition and sugar conditions beyond the conventional isotonic paradigm.