Synthesis of Compound Giant Unilamellar Vesicles: A Biomimetic Model of Nucleate Cells

A simple Giant unilamellar vesicle (sGUV) is a micron-sized spherical vesicle, which is composed of a lipid bilayer that encloses an aqueous solution inside and is suspended in another aqueous medium. As biomimetic models of biological cells, sGUVs are well-established systems for biophysical studies. However, sGUVs primarily mimic anucleate cells, limiting their application as models for non-nucleate cells. We propose compound Giant Unilamellar vesicles (cGUVs) as a more appropriate biomimetic model for nucleated cells. cGUVs are vesicle-in-vesicle structures, where the outer and inner vesicle bilayers can be assumed to represent the cell and the nuclear membrane of a cell, respectively. In this study, we describe a simple method for the synthesis of cGUVs. Briefly, sGUVs were generated using the lipid composition of DMPC and cholesterol within the range of 37-43 mol% in a sucrose medium via the electroformation method. These sGUVs were then subjected to osmotic shock by introducing a hypertonic glucose solution, triggering an immediate transition to a somatocytic shape. This process led to the formation of an intermediate state where the outer and inner vesicles remained connected through a neck, ultimately resulting in the development of vesicle-in-vesicle structure, referred to as cGUVs. Furthermore, it is known that GUVs prepared with higher cholesterol levels in the bilayer exhibit reduced ion permeability. This characteristic allows tuning of conductivity in the annular solution of a cGUV by adjusting the conductivities of the hydrating medium, the inner solution through hypertonic glucose, and the outer solution by adjusting of the dilution or the addition of conductive media, making the resultant cGUVs with controlled conductivities in the outer, annular, and inner regions, relevant for electric field studies. We propose this efficient and straightforward approach for synthesizing compound vesicles that can be used as a biomimetic model for eukaryotic, nucleated cells, advancing their application in biophysical research.