QuantGUV: Quantifying Encapsulation Efficiency of Small Molecules in Giant Unilamellar Vesicles.

Synthetic cells, constructed through the self-assembly of small molecules, are designed to mimic life-like behaviors by encapsulating functional molecules. For such synthetic cells to accurately replicate cellular reactions, it is critical that the concentrations of encapsulated molecules mirror those in living systems, as reaction kinetics and cellular network states are highly sensitive to these concentrations. However, current methods for precisely determining encapsulation efficiency in synthetic cells at the single-cell resolution have been limited. To address this challenge, we present QuantGUV, a software-driven, image-based analysis method that determines the concentrations of fluorescent molecules encapsulated within giant unilamellar vesicles (GUVs). We use QuantGUV to measure the encapsulation efficiencies of three fluorescent molecules, sulforhodamine B, mEGFP, and polystyrene beads for GUVs formed via the water-in-oil emulsion transfer method. The encapsulation efficiencies for polystyrene beads were close to 100% in most of the conditions, while sulforhodamine B and mEGFP's encapsulation efficiencies depended on the parameters during GUV formation, such as concentrations of lipids and oil-water ratio during GUV formation. By providing crucial insights into encapsulation efficiencies, QuantGUV offers a valuable tool to support the construction of quantitative synthetic cell systems with accurately controlled internal environments.