2.7. Characterization of Nanoparticles

The particle size and zeta potential of nanoparticle suspensions were determined by zeta potential and a particle size meter (Nanosizer, Malvern Instruments, Worcestershire, UK) and characterized using the dynamic light scattering (DLS) technique. Before the analysis, the samples were diluted 10 times with ultrapure water and the measurement process was carried out.

The morphological properties of dried nanoparticles were studied using a scanning electron microscope (SEM). The SEM (Zeiss EVO LS 10) has a magnification range of 20,000–50,000× and an accelerating voltage of 12 kV was used for the characterization of prepared RSG and CSG nanoparticles. All the samples were coated with gold before SEM analysis.

The membrane filtration technique was used to determine the encapsulation efficiency of NPs by total phenolic content using a Folin–Ciocalteu method. In the fabrication of NPs, after the ultrasonication process, nanoparticle suspensions were filtered with a 10 kDa membrane filter in a stirred cell (HP4750, Sterlitech, WA, USA) at 25 °C. After the filtration, non-encapsulated OPE was collected and used for TPC analysis to determine the actual amount of OPE encapsulated in NPs. The lyophilized OPE was prepared at a rate of 0.1–1 mg/mL and a calibration curve was plotted using total phenolic content (R² = 0.9843). The EE% of the OPE-loaded RSG and CSGNPs were calculated using the following equations:

The thermal properties of the CSG, RSG, OPE, RSGNP, CSGNP, and OPE-loaded RSGNP and CSGNP were evaluated by differential scanning calorimetry (DSC, TA Q20, DE, USA) equipped with a thermal analysis automatic program. Samples of 5 mg were placed in an aluminum pan with a capacity of 40 µL and sealed with an aluminum lid. An empty pan was used as a reference. Conventional DSC measurements were carried out by heating the sample at a rate of 10 °C/min 30 to 400 °C under a nitrogen stream.

The CSG, RSG, OPE, RSGNP, CSGNP, and OPE-loaded RSGNP and CSGNP were characterized by Fourier-transform infrared spectroscopy (FTIR) using KBr pellets in a spectrophotometer. FTIR (Bruker Tensor 27, MA, USA) characterization was performed in the transmittance mode, operating with wave numbers between 600 and 4000 cm⁻¹, using 32 scans with a resolution of 4 cm⁻¹.

The method of Tabasi et al. (2017) [16] was modified and used to perform the release of the OPE from RSG and CSG NPs. In vitro release of OPE from NPs was determined at 37 °C for physiological pH (7.4). Phosphate buffer saline (PBS) was used to mimic the physiological pH. OPE release from RSG and CSG NPs was determined using a dialysis method. Solutions of 40 mg of RSG and CSG NPs were weighed and transferred to the dialysis bag (14 kDa) with 5 mL of distilled water. Dialysis bags were sealed on both sides and then immersed in the gastrointestinal release medium (45 mL PBS) on a shaking incubator (WiseCube Fuzzy Control System, Germany) (100 rpm) at constant temperature (37 °C). The release medium was withdrawn (0.5 mL) for analysis and replaced with a fresh medium (0.5 mL) at different time intervals (0, 30, 60, 120, 240, and 1440 min). The release of OPE was evaluated using total phenolic content spectrophotometrically (Shimadzu UV-1800, Japan). The test was carried out in triplicate.

The total phenolic contents of the extract and NPs were determined according to the modified method described by Singleton et al. (1965) [34]. The total phenolic content was determined at 760 nm using a spectrophotometer (Shimadzu UV-1800, Japan) with Folin–Ciocalteu phenol reagent. The total phenolic contents of the samples were calculated as milligram gallic acid equivalent (GAE) per g dry sample. The cupric reducing antioxidant capacity (CUPRAC) analysis was carried out as previously described by Apak et al. (2004) [35]. Results were expressed as mg of Trolox equivalents per gram of dried OPE and dry weight of nanoparticles (mg TRE/g).