**3. Results and Discussion**

*3.1. Characterization of Hollow Zein Particles*

3.1.1. Effect of Na2CO3 and Chitosan on Hollow Zein Particles

The size of hollow zein particles is greatly influenced by the concentration of Na2CO3 used for the preparation of sacrificial templet [1,27]. The smallest particles of 76 nm were fabricated with 1% Na2CO3 (Figure 1A). At 0.5 and 2% Na2CO3, the size of HZ particles was 145 and 210 nm, respectively. The reason for the bigger particles is due to the formation of a thicker zein shell around all the available sodium carbonate nuclei formed in ethanolic conditions at a Na2CO3 concentration of 0.5% but the formation of bigger Na2CO3 nanocrystal in size due to excessive aggregation and crystal growth at 2% [27]. The pH values of the HZ particle dispersions prepared with 0.5, 1 and 2% Na2CO3 were 9.04, 10.31 and 10.77, respectively, which is above the pI of zein [28]. The ζ-potential of HZ particles was −8, −26 and −24 mV when prepared with 0.5, 1 and 2% Na2CO3 (Figure 1B), receptively. There was a slight increase in the particle size of HZ particles upon the addition of chitosan (Figure 1A), except that precipitation was observed at 0.05% chitosan and 2% Na2CO3. The increase in the particle size was the most obvious when the chitosan concentration was 0.1% at 1% and 2% Na2CO3. In the presence of chitosan, the pH of particle dispersions shifted to 4.0–4.5. Chitosan interacts with proteins below their pI through hydrophobic, electrostatic, van der Waals and hydrogen bonding [12,29]. The ζ-potential of HZ-chi particles ranged between +30 and +58 mV (Figure 1B). The amine groups in chitosan contribute to the positive surface charge of composite zein particles [30,31]. These results suggest the formation of a chitosan shell.

**Figure 1.** Particle size distribution (**A**) and ζ−potential (**B**) of hollow zein particles fabricated with 0.5% (black), 1% (red) and 2% (blue) Na2CO3 and coated with chitosan at various concentrations (Mark at the right). \* indicates unstable particles.
