3.2.4. Phase-Solubility Studies

The phase-solubility studies of cholesterol were performed by dissolving cholesterol (20 mg/mL) in methylene chloride, followed by solvent evaporation (0.3 mL per vial) under a flow of nitrogen in cylindrical vials. This left a very thin layer of cholesterol on the inner surface of the vials. Aqueous CD solutions were added to the vials, which were tightly sealed and heated in an autoclave (121 ◦C for 20 min) [57]. The vials were opened after equilibrating them at room temperature overnight and a small amount of solid cholesterol was added to each vial, which were again allowed to equilibrate at room temperature for an additional 6 days. The seeding with cholesterol was performed to reduce the possibility of complications caused by differences in the solid-state of cholesterol. Finally, the aqueous cholesterol suspensions were filtered through a 0.45 µm nylon membrane filter, and the filtrate was analyzed using high-pressure liquid chromatography (HPLC).

Quantitative determination of cholesterol was performed on a reversed-phase HPLC system from Merck-Hitachi (Darmstadt, Germany) consisting of an L 4250 UV-Vis detector operated at 203 nm, L 6200 A Intelligent pump, AS-2000A Autosampler, D-2500 Cromato-Integrator, and Phenomex Luna (Cheshire, UK) 5 µm C18 reversed-phase column (150 × 4.6 mm). The mobile phase consisted of methanol, acetonitrile, isopropyl alcohol, and tetrahydrofuran (50:25:25:0.1).

**Author Contributions:** Conceptualization, M.P.; methodology, M.P. and G.C.; data curation, M.P., T.L. and G.C.; formal analysis, M.P. and G.C.; investigation; M.P. and G.C.; writing—original draft preparation, M.P.; writing, reviewing, and editing, M.P., G.C., F.P.-D. and T.L.; supervision, F.P.-D. and T.L.; funding acquisition, F.P.-D. and T.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financially supported by the European Union grant no. MSCA-ITN-2017- 765441 (transMed); the German Research Council (DFG) grant no. PA1751/10-1; and the Faculty of Pharmaceutical Sciences, University of Iceland.

**Data Availability Statement:** No data reported.

**Acknowledgments:** The authors gratefully acknowledge CycloLab, Budapest, Hungary, for providing us with the fluorescent cyclodextrins used in the study.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Sample Availability:** Samples of the compounds are not available from the authors.
