In-situ observation of forming cell-sized liposome from dry lipid films

Department of Physics, Kyoto University, Japan* Institute of Materials Structure Science, High Energy Accelerator Research Organization, Japan**
Mafumi Hishida* ○Hideki Seto* Norifumi L. Yamada** K. Yoshikawa*

Phospholipid molecule is a main constituent of biomembranes. Due to its amphiphilic property, phospholipid molecules usually assemble into multi-lamellar vesicles in water. On the other hand, most of living cells are surrounded by single bilayers of phospholipids. Thus, the methods of creating cell-sized liposome (giant vesicles) are important to investigate model cell systems or for applications such as a drug delivery. However, the mechanisms of forming cell-sized liposome are not fully understood, and only practical recipes are known so far.
From this viewpoint, we have focused on the natural swelling method in which cell-sized liposomes are created spontaneously by a hydration of dry lipid films deposited on a solid substrate. We tried to observe the process of swelling of multi-stacked phospholipids bilayers by means of time resolved small-angle X-ray scattering, because the mean repeat distance d of lipid bilayers is about 60 Å, and the process usually occur within tens of seconds. All the experiments were done at BL40B2, SPring-8, JASRI, Japan.
The hydration of lipids in the liquid-crystalline phase was found to proceed in 3 stages. In the early stage until about 20 s after hydration, water molecules penetrate between lipid bilayers on a solid substrate, and in the intermediate stage (20-100 s), the lipid bilayers swell about 12 Å and reach a quasi-stable state. In the late stage, lipid bilayers gradually peel off from the stack to form liposome. On the other hand, in the case of a lipid in the gel phase, only the early and intermediate stages are observed. These behaviors correspond to the capacity for the liposome formation depending on the lipid phases. The kinetics of the late stage is discussed in terms of Kramers’ formulas.