2.6.4. Viscosity

Viscosity of nanoemulsions was measured at di fferent angular velocities at a temperature of 25 ◦C using the Brookfield synchro-Lectric viscometer (LVDVI prime, Middleborough, MA, USA).

### *2.7. Transmission Electron Microscopy (TEM)*

Structural morphology of nanoemulsion droplets was investigated with TEM (Tecnai 20, Philips, Holland, OR, USA) operated at 200 kV and of a 0.15 nm e fficient in comprehensive resolution. The bright field imaging technique with high magnification and di ffraction modes was used to examine the morphology and structure of the nanoemulsion globules [15]. TEM imaging was carried out by staining a drop of the nanoemulsion with phosphotungstic acid solution (2% *w*/*v*) and directly placing on the copper grids, subsequently dried at room temperature (25 ± 2 ◦C).

### *2.8. In Vitro Release*

The drug release studies were performed employing a vertical Franz di ffusion cell having an effective surface area of 1.13 cm<sup>2</sup> and simulated tear fluid (pH 7.4) as the dissolution medium [16]. Briefly, 1 mL of nanoemulsion (equivalent to 5 mg of moxifloxacin per ml) or drug solution (control) in the stimulated tear fluid was taken on a previously soaked cellophane dialyzing membrane (MWCO 12–14 kDa, Spectra/por ® Spectrum Laboratories Inc., Rancho Dominguez, Berkeley, CA, USA) that separates the donor and receptor cell. The entire assembly was kept on a thermostatically controlled water bath set at 37 ± 0.5 ◦C and receptor medium was stirred at 50 rpm. Aliquots of sample (1 mL) were drawn at regular time intervals (0.5, 1, 2, 3, 4, 5 and 6 h) and replaced with the same volume of fresh media. The samples were subsequently diluted and analyzed for moxifloxacin content by HPLC. The data were analyzed to determine correlation coe fficient (*r*2) and release kinetics using various mathematical models [17];


where *Q* represents quantity of drug released in time *t*, *Q*0 represents value of *Q* at zero time, *k* represents the rate constant, *n* represents the diffusional exponent, *a* represents the time constant and *b* represents the shape parameter. The correlation coefficient (*r*2) and the order of release pattern was calculated in each case.

### *2.9. Ex Vivo Permeation*

The permeation studies were carried out using the Franz diffusion cell with a standard setup previously used in our earlier study [18]. Briefly, optimized formulation (MM3) or control (commercial eye drops; Vigamox™) (equivalent to 5 mg of moxifloxacin per mL) was placed in donor compartment and simulated tear fluid (pH 7.4) in the receptor compartment. Between donor and receptor compartment, the isolated rabbit cornea membrane was placed. The temperature of the medium was maintained at 37 ± 0.5 ◦C by the circulation of warm water through the outer jacket. Samples were withdrawn at predetermined time intervals (up to 6 h) and the same volume of fresh medium was replaced. The withdrawn samples were diluted and analyzed by HPLC.
