2.2.5. In Vitro Release Studies

In vitro dissolution tests were performed according to the European Pharmacopoeia procedures dealing with solid oral dosage forms. In particular, the paddle apparatus (apparatus 2, Sotax AT7, Teknokroma®, Barcelona, Spain) with sinkers was set at 150 rpm and 37 ◦C, filled with 1 L of dissolution medium and working at sink conditions. Stomach physiological fluid was simulated by using a dissolution medium composed of HCl 0.001 M, while simulated intestinal fluid (SIF) was used with a phosphate buffer (pH = 6.8) without enzymes.

Hard gelatin capsules were subjected to the in vitro dissolution test. The capsules were prepared with 210 mg of each PZQ–VHS and PZQ–SEP interaction product, in which 35 mg corresponds to PZQ. Each of these interaction products (PZQ–VHS and PZQ–SEP) were prepared with the different solvents previously explained. Similarly, hard gelatin capsules containing 35 mg of PZQ were elaborated as reference.

During the in vitro dissolution tests, at predetermined time intervals, 5 mL of samples were withdrawn, the volume immediately replenished with fresh dissolution medium. The drug amount was quantified by High-Performance Liquid Chromatography (HPLC) after being filtered through 0.45 μm nitrocellulose membranes (Merck-Millipore®, Darmstadt, Germany). HPLC was performed in a 1260 Infinity II HPLC (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a quaternary pump, autosampler, column oven and UV-VIS diode-array spectrophotometer. The stationary phase was a Lichrospher® (100 RP-18, C18, 5 <sup>μ</sup>m, 15 cm <sup>×</sup> 3.2 mm) column (Merck Millipore®, Darmstadt, Germany). Isocratic conditions were used, the mobile phase formed by a mixture of H2O and CH3CN (35:65 *v*/*v*). The flow rate was set at 0.8 mL/min with a sample injection volume of 10 μL. PZQ was detected at 225 nm during the 5 min of the run time of the HPLC analysis. The software LC Open LAB HPLC 1260 (Agilent Technologies Inc.) was used to record and treat the chromatograms. The linear analytical method was obtained in the concentration range of 5 to 100 mg/L of PZQ in both dissolution media (correlation coefficients of 1 in both HCl 0.001 M and in SIF).

Subsequently, release data fitting was performed using kinetic models intended to describe the drug release from immediate or modified dosage forms [35–37]. These models, such as zero order, first order, cube root, Higuchi, Peppas, and Weibull, were used to fit experimental data obtained from in vitro dissolution studies. The fitting was carried out linearizing the equations. The choice of the best model to describe the dissolution/release process of the drug was based on the correlation coefficient (R2) criterium. A second evaluation criterium that is frequently used is called the Akaike Information Criterion (AIC) [38,39]. Herein, it is also considered.

#### 2.2.6. Solubility Studies

The solubility of PZQ, PZQ–VHSet and PZQ–SEPet, which was used as a reference for the rest of the interaction products, were obtained in both HCl 0.001 M and SIF (phosphate buffer at pH = 6.8, without enzymes). PZQ solubility was obtained by placing 30 mg of drug in 10 mL of HCl and SIF (supersaturated conditions). The solution was stirred at 37 ◦C for 24 h (thermostatic bath). Then, the solution was centrifuged, the supernatant filtered (nitrocellulose membranes, 0.45 μm, Merck Millipore®) and quantified by HPLC (Agilent Techonologies Inc.). The amount of dissolved drug, determined by the chromatography, corresponds to PZQ solubility in the corresponding medium. In order to demonstrate the solubility improvement of PZQ in the composites (PZQ–VHSet and PZQ–SEPet), drug solubility was determined with the very same procedure. Triplicate experiments were performed in all samples.

#### 2.2.7. Cell Culture

Cellular viability and cell cycle profiles were studied over a human colorectal carcinoma cell line HCT 116 (ATCC® CCL-247™, Manassas, VA, USA). HCT 116 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco™, Dublin, Ireland) supplemented with 10% *w*/*w* of Fetal Bovine Serum (FBS, Gibco™, Dublin, Ireland), 1% of Glutamax (BioWhittaker®, Cologne, Germany) and 1% of penicillin/streptomycin (BioWhittaker®). Cells were cultured in an incubator set at 37 ◦C and 5% of CO2.
