Quantitative study of the structure of multilamellar giant liposomes as a container of protein synthesis reaction

Liposomes are widely used as cell-sized compartments for encapsulation of biochemical reaction systems to construct model cell systems. However, liposomes are usually diverse in both size and structure, resulting in highly heterogeneous properties as microreactors. Here, we report the development of a strategy to investigate the internal structure of giant multilamellar vesicles (GMLVs) formed by the freeze-dried empty liposomes (FDEL) method as containers of an in vitro transcription/translation system. To evaluate the occurrence of the protein synthesis reaction in GMLVs, we designed a cascade reaction system in which a synthesized enzyme hydrolyzes the fluorescent substrate, and thus the space where the reaction takes place in liposomes becomes fluorescent. We found that only a part of the liposome was reactable and not the entire internal volume, i.e., the hydrolysis reaction took place in only a part of the fractured compartment volumes in GMLVs. Simultaneous measurement of the whole internal volume of the liposomes and the quantity of reaction product of more than 100 000 liposomes using a fluorescence-activated cell sorter (FACS) revealed that the distribution of reactable volume was proportional to the whole internal volume regardless of the liposome size, i.e., the relation between the quantity of whole and reactable volume in GMLV was found to be scale-free. This information would allow us to reduce the geometric parameters of GMLV for quantitative analysis of reaction kinetics in liposomes. The present measurement and analysis method will be an indispensable tool for exploring high-dimensional properties of a model cell system based on giant liposomes.