2.3. Equilibrium Solubility Measurement

The media preparation and equilibrium solubility measurement method has been applied in previous DoE studies [11,12,13].

The concentration of each stock solution has been designed to be 15 times greater than the upper limit concentration value required for the DoE, with the exception of oleate, for which only a 5 times concentration was possible (see Supplementary Materials, Tables S1 and S2).

Sodium taurocholate, monoglyceride, lecithin and cholesterol were weighed and transferred into a flask, then 2 mL of chloroform was added to dissolve all the solid material. A stream of nitrogen gas was applied in order to remove the chloroform and to ensure the formation of a dried film. Water was added to reconstitute the dried film, and the solution was mixed to obtain a homogenous suspension, transferred to a 5 mL volumetric flask, and brought to volume with water.

Sodium oleate (1.90 g) was weighed and placed into a 50 mL volumetric flask, dissolved in water with the assistance of gentle heating (37 °C) to aid dissolution, and then made up to volume with water and kept under heat to aid solubilisation.

A concentration of 0.3 M monosodium dihydrogen phosphate buffer was prepared by adding 20.4 g into a 500 mL volumetric flask and making up to volume with water. This was split into two, and the pH was adjusted to 5 and 7 using aqueous 0.5 M HCl or 0.5 M KOH.

Excipient solution: The appropriate amount of each excipient was weighed out (see Supplementary Material, Table S3), transferred to a 25 mL volumetric flask, and brought to volume with water, with the exception of chitosan, which was dissolved in 0.1 M acetic acid and mixed overnight with a magnetic stirrer under heat.

Individual experimental solutions were prepared following previous published protocols that have been demonstrated to successfully permit the determination of equilibrium solubility [11,12,13]. The solution was prepared by the addition of an excess amount (above the estimated solubility) of fenofibrate powder [13] to a centrifuge tube (15 mL Corning^® Centristar™ cap, polypropylene RNase/DNase free, non-pyrogenic) followed by the addition of each component of the simulated intestinal fluid media according to the run order generated by the DoE together with the excipient to be examined (see Table S3). After all of the media components were added, the pH was adjusted to 5, 6 or 7 according to the run order using 0.1 M HCl or 0.1 M KOH, and tubes were capped and placed on an orbital shaker (OS 5 basic Yellowline, IKA, Staufen, Germany) for 1 h, after which the pH was readjusted if required. The tubes were then shaken in a tube rotator for 24 h at 40 rpm at 37 °C. After 24 h, a 1 mL amount was taken from each tube, transferred to a 1.5 mL Eppendorf^® tube then centrifuged at 15,000 rpm for 5 min. Following centrifugation, 0.5 mL of the supernatant solution was transferred to an HPLC vial to analyse drug solubility using HPLC, as will be discussed below.