# Hydrothermal Preparation of Faceted Vesicles Made of Span 40 and Tween
## 1 Introduction  
A vesicle is a closed-system vesicle composed of a lipid bilayer membrane and is one of the molecular assemblies formed by lipids [1; 2; 3; 4; 5; 6]. The particle size ranged from several tens of nanometers to several hundred micrometers. Amphiphilic molecules are known to form vesicles such as phospholipids (liposomes) [1; 2; 3], surfactants (niosomes) [4; 5], and polymers (polymosomes) [6]. Since vesicles can encapsulate drugs in the internal aqueous phase and membrane, they are considered for various applications. For example, vesicles are vehicles that efficiently transport drugs into the body. Research is also underway for the application of detection assays based on the leakage characteristics of encapsulated substances, artificial cell systems, and new nanobioreactors. The search for and synthesis of lipid materials suitable for such applications is ongoing. In recent years, some studies using Sorbitan Monooleate (Span)/Polyoxyethylene Sorbitan Monolaurate (Tween), which is cheaper than phospholipids, have shown progress [7; 8; 9]. The Spans have a sorbitol group as the hydrophilic head and an acyl chain in the hydrophobic tail. Tween is a molecule in which an oligoethylene glycol group is ether-bonded to the hydroxyl group at the head of the Span. The basic physical properties of the Span/Tween system are well known. However, unique features of vesicle formation have recently been discovered [8]. For example, Span 20/Tween 20 forms spherical vesicles (50:50), tubularvesicles (100:0), and lens-like vesicles (80:20), whereas Span 40/Tween 40 forms spherical vesicles (50:50) and faceted vesicles (80:20) [8]. The number of acyl chains in Span 80 (one to four acyl chains) determines the curvature of vesicles [7]. Thus, the mixture ratio and chemical structure of Span and Tween affect the morphologies and surface properties of vesicles, which are related to their function [8; 9; 10]. Of the various morphologies of vesicles, the faceted vesicles have recently been brought to attention. The faceted vesicle is a polyhedral vesicle that contains membrane defects at the rim of the facets [11]. Such faceted vesicles can be formed from many different materials such as catanionic amphiphiles [12], block copolymers [13], phospholipids [11; 14; 15], and fatty acid/fluorinated fatty acid systems [16; 17], rather than nonionic surfactants such as Span 40/Tween 40 [8]. For example, the mixture of fatty acid and fluorinated fatty acid can yield faceted vesicles by adjusting the pH of the fluorinated fatty acid [16; 17]. Alternatively, a cooling of the temperature to below the phase-transition temperature results in a formation of gel-phase interdigitated membranes and a subsequent phase separation of a flexible rim and stiff facets to induce the faceted vesicles [11; 18]. This phase separation has been revealed in the molecular dynamics study. Accordingly, the phase separation of Span 40 and Tween 40 might result in the formation of faceted vesicles although the details for mechanism have not been reported. Conventionally, a hydration method is a well-known technique [9]. This method is usually combined with a freeze-thaw method to increase the size of the vesicles [1; 2]. A two-step emulsification method was used to prepare vesicles made of the Span/Tween system [7; 8; 9]. However, these methods are batch-type, require complicated operations, and have the problem of using large amounts of organic solvents. Therefore, we recently reported a new preparation method that combines the subcritical water-assisted emulsification method (SCW) and the solvent diffusion method (DS), which is called the SCW/SD method [10]. Here, subcritical water is water that maintains its liquid state at high temperature and high pressure. A conceptual illustration of the SCW/SD is shown in Figure 1. First, a coarse O/W emulsion (size 800 nm) was produced by emulsifying a three-component water/oil/surfactant mixture.