*2.3. Optimization and Statistical Design of LEO-NEs*

#### 2.3.1. Single-Factor Experiments (SFE)

Single-factor experiments were designed to investigate the effects of hydrophilic–lipophilic balance (HLB) value, content of Span-80 and Tween-80 (STmix), concentration of essential oil, ultrasonic time, and ultrasonic power on the mean droplet size, which can also provide a reasonable data range for the design of the response surface methodology. Specific parameters are presented in Table 1. The HLB value represented the combined affinity of hydrophilic and oleophilic groups in emulsifier molecules for oil or water [20]. Different HLB values of surfactants can contribute to the formation of two types of emulsions: water-in-oil (W/O) emulsion and oil-in-water (O/W) emulsion. To prepare the O/W LEO-NEs with hydrophilicity, an oil-in-water emulsifier with a high HLB value (8–15) was chosen. According to Nirmal et al. [21], different combinations of STmix were used to create surfactant HLB values ranging from 8–15, as shown in Table 2.

**Table 1.** Variables of single-factor experiments (SFE) and Response surface methodology (RSM).



**Table 2.** Different combinations of Span 80 and Tween 80 used to create surfactant HLB value.

#### 2.3.2. Response Surface Methodology (RSM) Design

The levels of the independent variables to be used in the Box–Behnken designs were determined by the results of the SFE. The RSM explored the effects of the selection factor over 29 runs. In this work, the BBD with four variables (factor A was the HLB value, factor B was the STmix content, factor C was the ultrasonic time, and factor D was the ultrasonic power) at three levels (−1, 0, 1) was carried out to evaluate the effect on the dependent variable. The mean droplet size (Y) was the response value. The optimum formulation was chosen by the analysis of the RSM. The specific parameters are shown in Table 1.
