## 2 Results and Discussion
### Characterization and In Vitro Diffusion Study of PEs
#### 2.1.3 Characterization of PEs  
Properties of PEs are influenced by many parameters, like the interfacial surface tension of the phases, size, wettability, and concentration of stabilizing particles, o/w phase ratio, and emulsification energy. In this study, we examined the influence of the o/w phase ratio and size of the stabilizing SNPs on the droplet size and stability of formulated PEs, while other parameters were kept constant.  
The results can be seen in Table 3, including data for composition, droplet size, and appearance of PEs. For the microbiological experiments and in vitro diffusion studies, we used emulsions that were stable for at least one week, which means that their droplet size did not change within this period, and creaming, phase separation, aggregation, or sedimentation of SNPs did not occur.  
As shown by [PERSON] and [PERSON] (2016), the size of stabilizing particles influences the emulsion droplet size at the same o/w phase ratio. We found that the increment of oil phase concentration caused an increment of emulsion droplet size at all examined oil phase concentrations. When 20 nm SNPs were used, the droplet size increased until reaching a droplet size of 1.8 \(\upmu\)m. Further increment of oil phase concentration did not cause significant droplet size increment, and the stability of emulsions was much higher in the 11.16-16.12 mg/mL concentration range (for oil phase) (see Table 3). We observed a similar effect when 50 nm SNPs were used in the 4.48-11.19 mg/mL concentration range and 1.6 \(\upmu\)m droplet size (Table 3). We could not observe such behavior for PE 100 ET. In this case, the droplet size continuously increased as the oil phase concentration increased, and the stability of emulsions was much lower (less than one week).  
The stability of PE was also influenced by the size of the stabilizing SNPs. We found that with the increasing SNPs size, the stability of PE decreased, at the same o/w ratio. The PE stability was 20 weeks using 20 ET, 8 weeks for 50 ET, and 1 week for 100 ET SNPs, at 11.19 mg/mL oil phase concentration. The zeta potential values of PEs could also provide their colloidal stability, whose values did not differ significantly from the SNPs suspensions. The zeta potential of 20 ET, 50 ET, and 100 ET SNPs was \(-\)28.3, \(-\)25.2, and \(-\)25.0 mV, while the values of PE 20 ET, PE 50 ET, and PE100 Et in the 0.90-17.91 mg/mL oil concentration range were from \(-\)28.0 to \(-\)19.3 mV, \(-\)24.9 to \(-\)19.0 mV, and \(-\)24.3 to \(-\)17.6 mV, respectively.  
The type of emulsions was determined by conductivity measurements. The conductivity values of stabilizing SNPs suspended in distilled water were 215.0, 211.4, and 268.3 \(\upmu\)S-cm\({}^{-1}\) for 20 ET, 50 ET, and 100 ET, respectively, while conductivity for the oil phase was 0.058 \(\upmu\)S-cm\({}^{-1}\). The conductivity values of PEs were in the 157.33-257.50 \(\upmu\)S-cm\({}^{-1}\) range, which means that all the PEs were o/w type emulsions.  
\begin{table}
\begin{tabular}{c c c} \hline
**Compounds** & **Retention Time t\({}_{\text{R}}\) (min)** & **Percentage Ratio of Compounds (\%)** \\ \hline \(\alpha\)-thujene & 5 & 1.7 \\ \(\alpha\)-pinene & 5.2 & 4.6 \\ \(\beta\)-phellandrene & 5.2 & 0.6 \\ \(\beta\)-pinene & 6.2 & 1.2 \\ \(\beta\)-mycrene & 6.4 & 0.8 \\ \(\alpha\)-terpinene & 7.0 & 1.4 \\ \(p\)**-cymene** & **7.3** & **35.2** \\ terpily-acetate & 7.3 & 2.1 \\ cineole & 7.4 & 5.8 \\ \(\gamma\)-terpinene & 8 & 7.6 \\ terpinolene & 8.6 & 1.7 \\
**terpinene-4-ol** & **10.7** & **32.5** \\ \(\alpha\)-terpineol & 11 & 2.6 \\ aromadendrene & 15.5 & 0.7 \\ epiglobulol & 16.4 & 1.2 \\ \hline \end{tabular}
\end{table}
Table 2: Composition of _Melaleuca alternifolia_ EO. The results of GC analysis showed the average of the two parallel measurements in the percentage of volatile compounds. The main components of MA have been highlighted.