*3.2. The Size and ζ-Potential Analysis of SeNPs and PSP-SeNPs*

The concentration of the polysaccharides is an important factor that influences the size of SeNPs, further affecting their functionality in food or medical application [30]. Thus, the effect of PSP concentrations on the hydrated particle size and the corresponding polydispersity index (PDI), as well as the ζ-potential of nanoparticles in the aqueous solution was investigated first. The particle size of barely SeNPs was up to 157 nm (Figure 2A). The addition of PSP at different concentrations could decrease the average size of SeNPs. The average diameter of PSP-SeNPs significantly decreased from 151 to 132 nm as the concentration of PSP increased from 0.01 to 0.075 mg/mL. PSP-SeNPs showed the smallest average size of 114 nm at the PSP concentration of 0.1 mg/mL, whereas further increases in PSP concentration from 0.125 to 0.25 mg/mL resulted in an increase in the size from 123 to 152 nm. It might be due to PSP at a low concentration was not enough to control the formation of SeNPs and prevent them from aggregation [31]. On the other hand, too high

PSP concentration represented more PSP chains coated on the surface of SeNPs, resulting in a larger hydration particle size [32]. As shown in Figure 2B, SeNPs in the absence of PSP exhibited a negative ζ-potential at −20.3 mV. The ζ-potential values of PSP-SeNPs were determined to be approximately −24.7, −26.6, −29.6, −30.4, −32.8, −34.9 mV at the PSP concentration of 0.01, 0.05, 0.075, 0.1, 0.125, 0.25 mg/mL. The absolute ζ-potential values of PSP-SeNPs increased with the PSP concentration increasing, further demonstrating that negatively charged PSP was exposed on the surface of SeNPs. Moreover, the higher magnitude of ζ-potential represents greater stability of nanoparticles [13], suggesting that the SeNPs decorated with PSP possess higher stability than barely SeNPs. PSP-SeNPs prepared by 0.1 mg/mL PSP were used in the following experiments.

**Figure 2.** Size distribution (**A**) and ζ-potential (**B**) of SeNPs and PSP-SeNPs prepared with different concentrations of PSP (0.01–0.25 mg/mL). Values marked with \*: *p* < 0.05, \*\*: *p* < 0.01, and \*\*\*: *p* < 0.001 indicated significant differences when compared to SeNPs.

## *3.3. Morphological and Structural Characterizations of SeNPs and PSP-SeNPs*

The morphology and size of SeNPs and PSP-SeNPs were further characterized by TEM. Figure 3A,B exhibited the TEM images of SeNPs in the absence of PSP. The results showed that adjacent SeNPs agglomerated together and presented a dendritic structure. The large-sized cluster and aggregates can also be easily visualized. However, the SeNPs in the presence of 0.1 mg/mL PSP (Figure 3C,D) exhibited a homogeneous and monodisperse spherical structure with an average size of about 105 nm, confirming the important role of PSP in regulating and stabilizing SeNPs. It should be pointed out that the hydrodynamic radius of the nanoparticles provided in the DLS analysis was larger than the size observed in the TEM image, which was sensitive to the electron-rich nanoparticles. The HRTEM image (Figure 3E) of an individual PSP-SeNPs showed a distinct lattice fringe with an interplanar spacing of 0.43 nm, revealing the excellent crystallinity of PSP-SeNPs. The elemental composition and distribution of the PSP-SeNPs were further determined by EDX. As shown in Figure 3F, the strong C, O, and Se element peaks were observed in EDX spectra. The PSP-SeNPs had a 63.10% weight percentage of C atom, together with 10.95% O atom and 25.94% Se atom. Furthermore, no other peaks for other elements were detected, indicating that PSP was successfully coated on the surface of SeNPs and confirming the purity of PSP-SeNPs [33].

**Figure 3.** TEM images of SeNPs (**A**,**B**) and PSP-SeNPs in the presence of 0.1 mg/mL PSP (**C**,**D**). HRTEM of an individual PSP-SeNPs (**E**) and typical EDX from HRTEM (**F**).
