2.2.5. Stability of SeCS

The stability of nanoparticles is one of the key factors for their biological function. Studies have shown that the stability of nanoparticles is closely related to pH, storage temperature and storage time in the application medium [24,25]. Therefore, we next investigated the influence of different pHs and storage temperatures on the stability of SeCS. The influence of pH on the stability of SeCS was shown in Figure 6A. The particle diameters of SeCS notably decreased to approximately 98.9 ± 5.9 nm at the 2–8 pH range. Then, no significant shift occurred at the 8–12 pH range. This was probably attributed to the strongest electrostatic interaction between anionic CS and SeNPs at pH 8.0 due to the sensitivity of CS at a low pH [26]. The effect of storage temperature on the stability of SeCS was shown in Figure 6B. When the SeCS solution was stored at 4 ◦C for 28 days, the particle size showed no obvious change. Yet, the particle diameter of the SeCS solution stored at 25 ◦C for 28 days significantly increased to 262.7 ± 13.6 nm. It was speculated that an increase in particle diameter might be related with the changes of the internal structure of the SeCS because the increasing temperature resulted in a change in the amorphous state of SeCS to the crystalline state. Moreover, our results were consistent with previous studies that high temperature was not conducive to the stability of selenium nanoparticles [10,26]. These results indicated that SeCS exerted excellent stability under a refrigerating temperature and alkaline environment.

**Figure 6.** Effects of different pHs (**A**) and storage temperatures (**B**) on the size of SeCS. The initial pH value of the control group was 6.8. *p* < 0.05 (\*) or *p* < 0.01 (\*\*) means that columns between control group and other groups are significantly different.

## *2.3. The Antioxidant Property of SeCS*

The antioxidant potential of SeCS was analyzed using the DPPH assay and ABTS assay in which ascorbic acid was used as a standard. As shown in Figure 7, the DPPH and ABTS radical scavenging rates of CS and SeNPs were extremely low. However, comparing with CS and SeNPs, the DPPH and ABTS radical scavenging rates of SeCS significantly increased. With the concentration of SeCS increased, the DPPH and ABTS radical scavenging rates of SeCS increased from 29.13 ± 3.28% (0.1 mg/mL) and 13.92 ± 2.57% (20 μg/mL) to 66.69 ± 2.71% (0.5 mg/mL) and 52.44 ± 2.29% (100 μg/mL), respectively. Our results indicated that SeCS could effectively scavenge the DPPH and ABTS free radical in a dosedependent manner. However, the DPPH and ABTS scavenging activities of SeCS were lower than the VC, particularly in the ABTS scavenging assay.

**Figure 7.** DPPH radical scavenging rate (**A**) and ABTS radical scavenging rate (**B**) of SeCS. *p* < 0.05 (\*) or *p* < 0.01 (\*\*) means that columns between SeCS at 0.1 mg/mL and SeCS at other concentrations are significantly different. *p* < 0.01 (##) means that columns between SeNPs at 0.1 mg/mL and SeNPs at 0.4 mg/mL are significantly different. *p* < 0.05 (&) or *p* < 0.01 (&&) means that columns between SeCS at 20 μg/mL and SeCS at other concentrations are significantly different. *p* < 0.05 (@) means that columns between SeNPs at 20 μg/mL and SeNPs at other concentrations are significantly different.
