*3.4. The Stability of SeNPs and PSP-SeNPs*

Stability is an important factor influencing the functionality and applications of nanomaterials. In this study, the effect of pH, temperature, and ionic strength on the stability of PSP-SeNPs was investigated. As shown in Figure 4A, the average size of PSP-SeNPs significantly decreased from 1262 to 186 nm when pH was increased from 2 to 3. It could be observed that no obvious changes occurred in the average size at pH range from 4 to 10. Similar results were also described previously on the stability of Polyporus umbellatus polysaccharide (PUP) coated SeNPs [34]. This might be ascribed to the protonation of PSP at pH 2 that weakened the electrostatic interactions between SeNPs and PSP, leading to the aggregation of nanoparticles. Moreover, the ζ-potential of PSP-SeNPs kept increasing with pH increased and reached the highest value of −32.6 mV at pH 7. A further increase in pH did not significantly affect the ζ-potential of PSP. It has been reported that the ζ-potential of nanoparticles was highly associated with the pKa value of the polysaccharides. The pH value higher than the pKa of polysaccharides resulted in more deprotonated characteristic groups, contributing to the increase in ζ-potential [27]. The average size of PSP-SeNPs increased from 113 to 191 nm, accompanied by the temperature increase from 25 ◦C to 90 ◦C with a constant ζ-potential at around −31 mV (Figure 4B). The result indicated that heating could increase the chances and strength of collisions, resulting in a larger size [29]. As shown in Figure 4C, the particle size of PSP-SeNPs exhibited a slight increase in 10 and 50 mM NaCl with decreased ζ-potential, and steeply increased to 882 nm in a high concentration of NaCl at 100 mM. High ion strength could remarkably reduce the surface

charge of nanoparticles due to the electrostatic interaction between positive charged Na+ and negatively charged PSP-SeNPs, resulting in the decrease of the electrostatic repulsion among nanoparticles [35]. It was observed that PSP-SeNPs were stable at about 113 nm for at least 20 days of storage (Figure 4D). The stability of PSP-SeNPs was higher than that of SeNPs decorated with a hyperbranched polysaccharide from Lignosus rhinocerotis 14. It should be pointed out that SeNPs in the absence of PSP precipitated after 1-day storage (Figure 1). Moreover, the particle size of PSP-SeNPs only increased from 113 to 123 nm after 30 days of storage and the ζ-potential of PSP-SeNPs presented at around −30 mV during the storage time, suggesting that PSP-SeNPs had better stability.

**Figure 4.** Effect of pH (**A**), temperature (**B**), ion strength (**C**), and storage time (**D**) on the average size and ζ-potential of PSP-SeNPs. Values marked with \*: *p* < 0.05, \*\*: *p* < 0.01, and \*\*\*: *p* < 0.001 indicated significant differences when compared to the conditions of pH: 7, temperature: 25 ◦C, NaCl: 0 mM, or storage time: 0 day.
