2.4. Drug Loading and Release

Loading of drug models was performed by absorption. Hydrogel samples (~20 mg) were immersed in a ratio of 2.2 mg/mL in the MB aqueous solution (0.1 mg/mL, at 37 °C) or in a curcumin solution in 3/2 v/v ethanol/water mixture (2 mg/mL, at 30 °C), respectively. After 6 h, the loaded samples were separated and freeze-dried, and maintained in dark conditions. The supernatant was subjected to spectrophotometric analysis of the unabsorbed drug to quantify the amount of loaded drug, with reference to appropriate standard curves.

The in vitro delivery study was carried out in phosphate buffer 0.01 M with a pH of 7.4. Briefly, dried loaded hydrogel pieces of about 20 mg were placed in 9 mL PBS solution, and maintained at 37 °C with gentle periodical shaking. At predetermined time intervals, aliquots of 2 mL were extracted and replaced with equal volumes of fresh PBS solution. The released drugs were quantified spectrophotometrically. For curcumin content quantification, the supernatant samples were completed with ethanol to obtain 3/2 v/v ethanol/water solutions, and eventually diluted. Experiments were performed in triplicate.