*2.14. Data Analysis*

A plot of the cumulative amount of moxifloxacin permeated across the cornea was plotted against time and the slope was measured as flux [23]. The statistical evaluation of the data was analyzed using one-way analysis of variance (ANOVA) (SPSS 23, SPSS Inc., Chicago, IL, USA). The statistical di fferences between values showing *p* < 0.05 were considered as significant.

### **3. Results and Discussion**

### *3.1. Pseudoternary Phase Diagram*

Preliminary studies were carried out to select ingredients for preparing moxifloxacin nanoemulsion. Indeed, the selection of the vehicle is extremely important as it provides good solubilizing e fficiency of the drug, which is essential for constituting a nanoemulsion [24]. Hence, the solubility of moxifloxacin in various oils, surfactants, and cosurfactants was determined by the standard procedures. Based on the high moxifloxacin solubility, ethyl oleate (oil; solubility ~28.37 mg/mL), Tween 80 (surfactant; solubility ~10.56 mg/mL) and Soluphor P (cosurfactant; solubility ~7.22 mg/mL) were selected for nanoemulsion preparation. The utility of the selected vehicles in preparing nanoemulsions is described in the literature. For instance, ethyl oleate has been used in nanoemulsions due to its lower molecular size (310.51 g/mol) as related to medium chain triglycerides (~800 g/mol). Furthermore, nanoemulsions formulated with ethyl oleate have demonstrated enhancement in corneal permeation of drugs [25]. Similarly, Tween 80 (up to 10% *w*/*w*) has been used as a surfactant in many commercial ophthalmic preparations which usually do not cause ocular irritation [26]. Due to the longer hydrocarbon chain length of Tween 80, it can broaden the area of the nanoemulsion region [27]. In addition, Soluphor P has demonstrated improved drug penetration across the biological membranes [28].

The pseudoternary phase diagram of the systems consisting of surfactant: Cosurfactant (Tween 80: Soluphor P) mixture (*S*mix), oil phase and water was illustrated in Figure 1A. The binodal curve separating two phase and one phase in the pseudoternary phase diagram was indicated by the visual observation of the sample appearance from turbid to transparent or vice versa. To complete the entire nanoemulsion domain, in addition to the water titration method, oil and water components were fixed and surfactant component varied. Similarly, water and *S*mix components were fixed and the oil component varied. The nanoemulsion domain obtained by these trials at ratios of 1:1, 1:2 and 2:1 with surfactant (Tween 80) to cosurfactant (Soluphor P) ratio were plotted in the phase diagram (Figure 1A). The phase behavior study revealed that, when the surfactant to cosurfactant ratio was 1:2, the maximum quantity of oil can be included in the nanoemulsion system. From the phase diagram, it was indicated that the change in phase behavior within the nanoemulsion region is mainly due to the hydrophobic carbon chain length of the oil and the ratio between surfactant and cosurfactant mixture used in the formulation. Therefore, it is likely that the hydrocarbon chain length compatibility among surfactant and oil is an important factor that a ffects the globule size formation and stability of nanoemulsion as suggested by Schneider et al. [29].
