Background

Liposomes are spherical vesicles composed mainly of double-layered phospholipids, similar to a living cell membrane; thus, they have been used as containers for biomolecules to construct artificial cells that allow for biochemical reactions (e.g., gene expression) in the interior separately from and/or in communication with the exterior (Walde et al., 2010).

Several methods are available for preparing liposomes that encapsulate biomolecules. In particular, a manual emulsion transfer method is useful for readily preparing relatively large liposomes with a diameter of more than 1 μm (called giant unilamellar vesicles, GUVs) without the need for special equipment such as an evaporator or a microfluidic device (Pautot et al., 2003; Noireaux et al., 2004). In this method, a water-in-oil emulsion that is covered with a phospholipid layer in an upper oil phase (containing phospholipids) is transferred into a lower water phase to be further covered with another phospholipid layer in the phase interface, thus forming a liposome.

Although a centrifuge is generally used for the phase transfer, an emulsion droplet can be transferred spontaneously just by adding a high-density solute (e.g., sucrose) without centrifugation, which often forces the formed liposome to burst (Hamada et al., 2008). Needless to say, the difference in density between the droplet and the water phase is key to the successful spontaneous phase transfer. This spontaneous method enables us to control the size of a liposome by adjusting the droplet volume, and surprisingly, to prepare a supergiant liposome with a diameter of more than 1 mm (called a supergiant unilamellar vesicle, SGUV), which can be observed by the naked eye without a microscope using a microliter-sized droplet (Kubatta et al., 2009).

However, conventional protocols for spontaneous emulsion transfer have the drawback that they require the inclusion of a considerably high concentration of sucrose (10-30% w/v) in the droplet that is to be spontaneously transferred. Such high amounts of sucrose severely inhibit gene expression, one of the most important biochemical reactions (Elani et al., 2015). For example, eukaryotic cell-free translation based on wheat germ extract (WGE) is suppressed by more than 80% in the presence of 10% w/v sucrose (Takahashi and Ogawa, 2020). Thus, we optimized the preparation conditions to permit droplet transfer at a much lower concentration of sucrose (approx. 1.5% w/v) that hardly affects WGE translation (Takahashi and Ogawa, 2020).

Our protocol with the optimized conditions allows us to prepare WGE-encapsulating SGUVs in which WGE translation takes place as efficiently as outside a liposome. The average successful formation rate of SGUVs is more than 90%, and the formed SGUVs are mostly stable for more than 60 min. The size of SGUVs can be controlled precisely (diameter = 1.6-4.4 mm) without impairing their formation or stability just by changing the volume of the droplet. No special equipment is required for the preparation or observation of SGUVs. Moreover, given that a WGE-based translation system enables the expression of a broad range of genes from various types of organisms (both eukaryotes and prokaryotes) as well as viruses (Madono et al., 2011), WGE-encapsulating SGUVs prepared using our protocol would be useful as a versatile research tool in cell-free synthetic biology (Hodgman and Jewett, 2012; Lentini et al., 2016; Kai and Schwille, 2019).