## 3 Results and Discussion  
### Preparation and Particle Characterizations of ZnSO\({}_{4}\)/SP NCs  
In this study, the homogeneous dispersion of inorganic molecules (e.g., ZnSO\({}_{4}\)) in SP by HME processing with twin screws was prepared. A twin screw extruder, compared to single screw extruder, provides the following advantages: a higher dispersibility, easier feeding, a lower risk of overheating, higher process productivity, and a better modulation of process parameters [9; 19; 20].  
Strong forces produced by twin screw systems can disperse ZnSO\({}_{4}\) homogeneously in an SP matrix and HME processing with SP can reduce the surface energy and prevent the occurrence of aggregates. Compared to micro-size particles of ZnSO\({}_{4}\), nano-sized particles of ZnSO\({}_{4}\) have the following advantages: the feasibility of intravenous administration, efficient cellular entry, and improved absorption across the mucosal membrane. HME has been used for preparing solid dispersion formulations of active pharmaceutical ingredients [9,21]. Molecular dispersing during the HME process can transform the crystalline property of the drug to an amorphous state, and it may contribute to the improvement of dissolution and bioavailability of the drug [9,13,14,22]. In addition, HME processing of a hydrophilic drug can provide taste masking of the drug [9,23]. Although HME formulations of organic substances have been widely developed, there are a few reports regarding the inorganic materials-based carriers prepared by HME processing [24].  
The mean diameter of ZnSO\({}_{4}\) dispersion in DW was greater than 5 \(\upmu\)m in this study. In this investigation, SP was used as a polymer matrix (host), so its feeding content was designed to be higher than ZnSO\({}_{4}\) (guest molecule). In our preliminary study, the mean diameters of formulations with several weight ratios between ZnSO\({}_{4}\) and SP were measured. The suggested ratio (ZnSO\({}_{4}\):SP = 3:7) was finally selected due to the smallest mean diameter of the prepared NC dispersion. It is expected that nano-sized carriers can improve the cellular entry and mucosal permeability of ZnSO\({}_{4}\). HME has been applied to prepare nanocarriers of ZnSO\({}_{4}\) in this investigation. The rotating screws force ZnSO\({}_{4}\) and SP towards the die, and those materials can be blended homogeneously and softened during the extrusion process (Figure 1). Pressure and heat during the HME process can disperse ZnSO\({}_{4}\) molecules homogeneously in SP. The amphiphilic property of SP might contribute to the fabrication of nano-sized ZnSO\({}_{4}\) particles. After the HME process, the extrudate was further pulverized and dispersed in aqueous media. As shown in Table 1, the mean diameter of ZnSO\({}_{4}\)/SP NC dispersion in DW was 75 nm. Considering its polydispersity index value (0.10 \(\pm\) 0.01), it can be concluded that the size distribution of NC dispersion is narrow. The zeta potential value of ZnSO\({}_{4}\)/SP NC dispersion in DW was close to zero. A similar mean diameter of ZnSO\({}_{4}\)/SP NCs was observed at pH 1.2, while a small portion of aggregates has been formed at pH 6.8 (Figure 2A). The polydispersity index values of ZnSO\({}_{4}\)/SP NCs at pH 1.2 and 6.8 were 0.04 \(\pm\) 0.01 and 0.17 \(\pm\) 0.01, respectively. They indicate the narrow size distribution of ZnSO\({}_{4}\)/SP NC dispersion at pH 1.2 and 6.8. The round shape of ZnSO\({}_{4}\)/SP NCs was also presented in TEM images (Figure 2B). The content of Zn in ZnSO\({}_{4}\)/SP NCs, measured by ICP-OES, was 11.44 \(\pm\) 0.55%, and its entrapment efficiency in NCs was approximately 100%. This implies that ZnSO\({}_{4}\) was successfully incorporated in the NCs during the HME process.  
Figure 1: Schematic illustration of the HME process for the development of ZnSO\({}_{4}\)/SP NC formulation.