3.3.2. pH Stability

Protein-based particles are liable to aggregation around pI [6], limiting their application. Quercetin-loaded hollow zein particles were sable with similar size distribution at pH 2, 3 and 4 (Figure 6A) but precipitated at pH 5 around pI, due to charge neutralization and lack of repulsive forces [50,51]. It is difficult to re-disperse the precipitated zein particles above the pI of zein. Quercetin-loaded hollow zein-chitosan particles showed two size distributions above and below pH 4 (Figure 6A). The change in the particle size below pH 4 is attributed to swelling and dissolution of the polymers with higher charge density [18], while the formation of bigger aggregates is at pH 5, close to the pI of zein [7]. The particles with chitosan were unstable at pH 6–9 due to the deprotonation of chitosan amine groups at pka and above [52,53]. It is noted that quercetin-loaded HZ-chi/pec particles showed good stability across the investigated pH range of 2–5 (Figure 6A). The ζ-potential of quercetin-loaded HZ-chi/pec was the highest at pH 5 and decreased as pH decreased (Figure 6B), due to deionization of the carboxylic groups of pectin below its pka (3.5) [16]. At pH 6–9, the size of quercetin-loaded HZ-chi/pec particles was around 220 nm and less than those at lower pH. Their ζ-potential also decreased slightly as the pH increased from pH 5. Karim et al. also reported a decline in the ζ-potential and size of pectin/chitosan-coated nanoliposome at pH 8 compared with that of pH 5 [18]. These changes might be due to the detachment of loosely adsorbed pectin molecules from the surface, since chitosan possesses less charged groups as the pH increases [54]. These findings suggest that the double coating with chitosan and pectin provides excellent protection against aggregation, especially around the pI of zein and pka of chitosan.

**Figure 6.** Size distribution (**A**) and ζ−potential (**B**) of quercetin−loaded hollow zein particles without (blue) and with chitosan (red) and chitosan/pectin (black) coating at various pH values.
