2.4.2. Ionic Strength Influence

Each nanoparticle dispersion was blended with NaCl to obtain samples with 0, 25, 50, 100, 150 and 200 mM NaCl concentrations and stored for 24 h for observing physical stability.

Size changes within nanoparticle dispersions were recorded using DLS at 25 ◦C.

#### *2.5. Encapsulation of 7,8-DHF into Ternary Nanoparticles*

7,8-DHF encapsulation was conducted according to the methods described in Section 2.2. 7,8-DHF, sophorolipid, and zein were dissolved at a mass ratio of 1:10:10 and 1:5:5 in 80% ethanol/water solution, respectively. The mass ratio of zein to polysaccharide was 5:1 in the final reaction system. 7,8-DHF encapsulation in binary and ternary nanoparticles were denoted as DHF-S/Z, DHF-CMC/S/Z, and DHF-ALG/S/Z. Loaded complex particles were reserved at 4 ◦C, with other samples freeze-dried for 48 h to conduct further analysis.

#### *2.6. Encapsulation Efficiency (EE) and Loading Capacity (LC)*

The EE and LC of encapsulated 7,8-DHF were assessed by UPLC based on our previously described method [25]. Specifically, the nanoparticles were centrifuged at 10,000× *g* for 10 min at 4 ◦C using a centrifugal machine (3K15, Sigma, Osterode, Germany). The supernatant (containing loaded 7,8-DHF) was removed and diluted 5-fold with methanol. And an equal volume of the initial suspension was diluted in 5-fold methanol to obtain initial 7,8-DHF. Then, EE and LC were calculated according to the following equation:

$$\text{EE (\%)}=\text{loaded 7.8-DHF/initial 7.8-DHF}\times 100\tag{1}$$

$$\text{LCC}\left(\%\right) = \text{loaded 7.8-DHF/weight of vehicle} \times 100\tag{2}$$

#### *2.7. 7,8-DHF Loaded Ternary Nanoparticles Characterization*

## 2.7.1. Fourier-Transform Infrared (FTIR) Spectroscopy

7,8-DHF and lyophilized sample under analysis were prepared by adding 99% KBr disc and scanned on an FTIR spectrometer (Avatar 370, Nicolet, Madison, WI, USA). The spectral scanning range was 500~4000 cm<sup>−</sup><sup>1</sup> at a resolution of 4 cm<sup>−</sup>1. The analytical results were carried out by OMNIC software version 8.0.

## 2.7.2. Circular Dichroism (CD) Spectrum

Secondary structural characteristics of nanoparticle dispersions under analysis were measured using a CD spectrometer (J-1500, JASCO, Tokyo, Japan). The concentration of complex dispersions was 0.2 mg/mL corresponding to zein. The buffer in CD experiments was deionized water (pH = 7). The secondary structure scanning region was 190~260 nm with a 0.1 cm path length. The bandwidth was 1.0 nm and scanning speed was 50 nm/min. The data were evaluated by Spectra Manager ™ II Software equipped with CD spectrometer.

#### 2.7.3. Differential Scanning Calorimetry (DSC)

Thermal behavior of 7,8-DHF and freeze-dried samples were studied via DSC (Mettler Toledo, Zurich, Switzerland). 2~10 mg of samples were accurately weighed and hermetically sealed in aluminum pans. An empty crucible under the same condition was used as a reference. Scanning calorimetry was performed from a range of 25 to 200 ◦C in N2 atmosphere at a heating rate of 10 ◦C /min under 30 mL/min flow.
