X-ray computed tomography (X-ray CT) is a noninvasive three-dimensional (3D) imaging technique used to diagnose various diseases. Contrast agents are used to increase the contrast of images acquired using X-ray CT. Currently, iodine-based contrast agents are often utilized because they allow for clear contrast as they are derived from iodine, which has a high atomic number (Z = 53) and relatively low toxicity to the human body. In general, small-molecule iodine-based contrast agents are known to have very rapid clearance from the blood [1], [2]. Therefore, several tens of milliliters to one hundred milliliters of contrast agent is often required per injection to provide sufficient contrast to the tissue to be diagnosed and further repeated dosing is necessary to obtain diagnostic images over time [1], [3], [4]. Further functionalization of small-molecule iodine-based contrast agents have a practical impact not only on improving the accuracy of X-ray CT diagnosis with iodine-based contrast agents but also on improving the quality of life of patients.

To address the issues surrounding dosage, various materials have been developed to improve the performance of iodine-based contrast agents [1], [2], [5], [6], [7]. Many efforts have been made to utilize iodine-based contrast agents more effectively by increasing the local concentration of iodine via encapsulation in nanosized carriers and conjugation with polymers, prolonging blood residence time and adding a targeting function to the affected area. Furthermore, these approaches are effective in reducing the serious side effects of contrast agents, such as allergic reactions and contrast-induced nephropathy. Encapsulation in micelles [8], [9], [10], [11], nanoemulsions [7], [11], [12], [13], [14], [15], lipid vesicles (liposomes) [7], [11], [16], [17], [18], [19], [20] and iodinated polymers (including dendrimers) [7], [11], [21], [22] has been reported. Among these, encapsulation in lipid vesicles is advantageous in the following respects:

(1) Lipid vesicles have a lipid bilayer structure similar to that of the cell surface, which makes them excellent biocompatible carrier materials.

(2) By encapsulating the contrast agent in a nanosized compartment (lipid vesicle), a higher local concentration can be maintained inside the lipid vesicles than the external concentration.

(3) Modification of lipid vesicles with hydrophilic polymers, such as polyethylene glycol, can suppress their uptake by the reticuloendothelial system (RES), such as the liver and spleen, preventing the recognition of lipid vesicles as foreign substances in the blood and significantly increasing their residence time in the blood [23], [24], [25], [26].

(4) It can facilitate the accumulation of contrast agents in specific pathological tissues and active targeting by incorporating reactive functional groups on the surface of lipid vesicles [27], [28], [29].

As noted by Tilcock [30], the technical challenge of lipid vesicular (liposomal) iodine-based contrast agents lies in their manufacturing processes, although their diagnostic efficacy has been proven in various studies. The encapsulation of water-soluble components in lipid vesicles is often significantly less efficient than the incorporation of lipophilic components. For example, in the lipid thin-film hydration method (commonly known as the Bangham method) the volume fraction of the internal aqueous phase of lipid vesicles in the total sample volume is very small and the to-be-encapsulated compounds must be randomly (non-selectively) entrapped in the internal aqueous phase of the lipid vesicles. To date, various methods such as reversed-phase evaporation [31], dehydration-rehydration [32], remote loading [29], [33], microencapsulation [34], [35], [36], [37], [38], inverted emulsion [39], [40] and microfluidic methods [19], [41], [42] have been investigated for encapsulating water-soluble compounds in lipid vesicles. The authors have recently developed a novel method for preparing lipid vesicles (the "multiple emulsification-solvent evaporation" method) based on a two-step emulsification technique [43], [44], [45] and have demonstrated that it can achieve high encapsulation efficiency for water-soluble components. The method also enables size control of lipid vesicles, which is important for intravenous administration of lipid vesicles [46], [47], [48]. In addition, the method for preparing lipid vesicles using double emulsions as the base material allows the incorporation of PEGylated lipids and various reactive functional groups [38], and thus which are expected to provide good blood retention and targeting properties. Therefore, the application of this method for the encapsulation of iodine-based contrast agents may provide important insights into the practical use of lipid vesicular contrast agents.

In this study, the encapsulation characteristics of a nonionic iodine-based contrast agent, iohexol (Ihex: molecular weight = 821.1 g/mol, iodine content = 46%, log P = −2.71 [49]), in lipid vesicles were investigated using a multiple emulsification-solvent evaporation method. In particular, the effect of the process parameters during lipid vesicle preparation on the efficiency of Ihex encapsulation was evaluated experimentally. The findings of this study are expected to improve the efficiency of X-ray CT diagnosis using iodine-based contrast agents.