2.3. Preparation of Chitosan Nanoparticles (CSNPs) Loaded with PPE

CSNPs were prepared by ionic gelation method using STPP as a crosslinking agent. LMW chitosan (0.2 g) was added to 40 mL acetic acid (1% v/v) to achieve a 0.5% w/v concentration, and it was then kept overnight under magnetic stirring at ambient temperature to get a clear solution, and its pH was adjusted to 4.6 using a 1 M NaOH. Chitosan solution was then filtered through a 0.45 µm syringe filter. PPE at different loading contents was added to the CS solution according to formulations shown in Table 1.

Different formulations (treatments) to prepare chitosan nanoparticles (CSNPs) incorporating pomegranate peel extract (PPE).

Chitosan solution containing PPE was magnetically stirred (at 500 rpm) for 60 min at ambient temperature (23 °C). A stock solution of STPP was prepared in double-distilled water to achieve a concentration of 0.2% (w/v); it was then filtered through a 0.22 µm syringe filter, its pH was adjusted to 5.6 by 1 M HCL, and it was stored at 4 °C before use. Chitosan nanoparticles were spontaneously produced by gradually adding 10 mL of cold STPP solution using a syringe pump (with a flow rate of 0.25 mL/min) into 10 mL of chitosan solution containing PPE under vigorous stirring (1400 rpm) for 50 min while holding in an ice-bath. The obtained suspension was then centrifugated at 9000 rpm for 30 min [29]. The supernatant was taken for subsequent analysis, and sedimented CSNPs were collected as pellets and washed several times by deionized water. The wet pellets were then subjected to probe sonication (VCX 130, Vibra Cell Sonics, Newtown, CT, USA) at 130 W/20 kHz/80% amplitude for 20 min (sonication/resting cycles of 1 min) in an ice-bath to get homogenous suspensions. CSNPs were then lyophilized at −60 °C for 72 h and kept refrigerated for further analysis.