3.5.1. FTIR

FTIR is a versatile tool for monitoring changes within the functional groups of biopolymers and evaluating intermolecular interactions between components and particles. As shown in Figure 4A, the spectrum of CMC and ALG included diversiform representative carbohydrate peaks. The broad 3446 and 3441 cm<sup>−</sup><sup>1</sup> peaks represented hydroxyl groups (O-H) stretching, and the sharp 1604 and 1615 cm<sup>−</sup><sup>1</sup> peaks are associated with carboxyl (-COO-) symmetrical stretching vibration [38]. The featured peaks of CMC and ALG spectra appear at 1418 and 1417 cm<sup>−</sup>1, corresponding to rhamnogalacturonan moiety [39]. Multiple simultaneous vibration peaks in CMC and ALG at 900~1350 cm<sup>−</sup><sup>1</sup> were accredited to characteristic peaks of polysaccharides [24]. In our previous study, we confirmed that there are electrostatic attractions, hydrophobic interactions, and hydrogen bonding in S/Z. The FTIR spectrum of S/Z nanoparticles showed typical characteristic peaks at 3368, 1657, and 1545 cm<sup>−</sup>1, respectively. When CMC and ALG were added, the peaks of O-H stretching vibration (3100~3500 cm<sup>−</sup><sup>1</sup> peaks) shifted from 3368 (S/Z) to 3382 (CMC/S/Z) and 3402 cm<sup>−</sup><sup>1</sup> (ALG/S/Z) [40]. This characteristic peak migration implied that there was a strong hydrogen bond between -OH groups in polysaccharides and amide group of glutamine in zein [41]. According to Liu et al.'s report [42], the 1657 cm<sup>−</sup><sup>1</sup> peak of zein at was the C=O stretching (amide I). 1545 cm<sup>−</sup><sup>1</sup> peak was primarily associated with bending of N-H coupled with the stretching of C-N (amide II). With CMC and ALG incorporation, the amide I and amide II characteristic peaks of ternary nanoparticles were switched to (1650 and 1651 cm<sup>−</sup>1) and (1544 and 1544 cm<sup>−</sup>1), respectively. These results revealed electrostatic attractions were related to the establishment process of CMC/S/Z and ALG/S/Z. Based on these results, we confirmed that ALG/S/Z possessed stronger hydrogen bonding and electrostatic attraction than CMC/S/Z. As seen from Figure 4B, the peaks at 3114, 1626, 1575, 1405, 1195, and 1071 cm<sup>−</sup><sup>1</sup> were the typical peaks of 7,8-DHF, which have been confirmed in our previous study [25,43]. Expectedly, these characteristic peaks of 7,8-DHF were vanished in both binary and ternary nanoparticles, indicating that DHF-S/Z, DHF-CMC/S/Z, and DHF-ALG/S/Z samples successfully encapsulated for 7,8-DHF.

**Figure 4.** FTIR spectra of CMC, ALG and bare complex nanoparticles (**A**), free 7,8-DHF and 7,8-DHF loaded in each complex nanoparticles (**B**), CD spectra of zein in DHF-Z, DHF-Z/S-CMC, and DHF-ALG/S/Z (**C**), DSC analysis of free 7,8-DHF, CMC, ALG, S/Z, and 7,8-DHF loaded each complex nanoparticles (**D**), XRD spectra of free 7,8-DHF, polysaccharides and loaded each complex nanoparticles (**E**).
