## 2 Materials and Methods  
Surface pressure/area (\(\pi\)-A) isotherms were measured [5] by Langmuir surface balance \(\mu\) Trough XS, area 135 cm\({}^{2}\), volume 100 mL (Kibron, Helsinki, Finland), via the Wilhelmy wire probe method (instrumental accuracy 0.01 mN/m). Phosphate-buffered saline (PBS, pH 7.4) was utilized as a trough subphase. Human meibum from healthy volunteers was collected by the soft squeeze method [1; 2] with permission from the institutional review board of Kyoto Prefectural University of Medicine, in agreement with the tenets of the Declaration of Helsinki and after the donors signed informed consent. The sample used was a pooled equiweight mixture of the MGS of four volunteers: two males (40 years old and 27 years old) and two females (41 years old and 28 years old). Until its use in experiments, the MGS solution (1 mg MGS/mL CHCl\({}_{3}\)) was stored at \(-\)80 \({}^{\circ}\)C. The mean molecular weight Mw = 700 (an average of 650 for wax esters and 750 for cholesteryl esters and (O-acyl)-omega-hydroxy fatty acids) was assumed for MGS [4].  
Rohto Dry Aid ophthalmic nanoemulsion (Table 1) and menthol were provided by Rohto Pharmaceutical, Osaka, Japan.  
An acrylic cover was fitted over the apparatus to protect the surface from dust and to suppress the evaporation of the PBS subphase (under the cover >90% relative humidity is maintained). A Hamilton micro-syringe was used to uniformly deposit human MGS at the air/water surface of the trough. After 15 min were provided for chloroform evaporation, dy  
\begin{table}
\begin{tabular}{c c} \hline
**Active Ingredients 1 ** & **Oil Phase ** \\ \hline \hline  & Sesame Oil/Menthol/Polyoxyethylene 10 \\  & Propylene Glycol (3.9 \(\times\) 10\({}^{-2}\) M) & Castor Oil/Polyoxyl 40 Stearate/Poloxamer \\  & 407 = 1.15/0.12/3.2/4.6/0.04 \\ \end{tabular}
\end{table}
Table 1: RDA composition as provided by the manufacturer. The active ingredient concentration is specified in brackets. The oil phase and the active ingredient aqueous phase are part of patent-protected TEARSHIELD TECHNOLOGYTM. All the components are dissolved in a pH 7.4 saline solution.  
namic compression-expansion isocycling of the film area was initiated by two symmetrically moving barriers at the maximum barrier's rate (70 mm/min), at which there was no leakage of the film. Five consecutive cycles were performed with each film studied. The \(\pi\)(A) curves attained constant shape after the third cycle and those \"stationary\" \(\pi\)(A) isotherms were analyzed further. All isotherms were repeated in triplicate with less than a 2% difference between the repetitions. The experiments were carried out at 35 \({}^{\circ}\)C, i.e., the physiological temperature of the ocular surface. The morphology of the films was monitored by a Brewster angle microscope (MC-BAM, Imperx, Boca Raton, FL, USA).  
The MGS film dilatational viscoelasticity was accessed by analysis of the surface pressure relaxation transients after a small rapid compression step was exerted on the layers [17; 18; 19]. In these experiments, after the film was equilibrated at initial surface pressure, \(\pi_{0}\) = 20 mN/m, a small compression step, \(\Delta\)A/A\(\omega\) = 5% \(\pm\) 1% (where Ao--initial film area, \(\Delta\)A--area change) was applied to it. The \(\Delta\pi\) relaxation transients were collected and analyzed further.  
For visualization of MGS spread, alone and in the presence of RDA oil phase (obtained via chloroform extraction from RDA), the air-water surface of the trough surface is covered with talc particles and the rate and degree of spread are visualized by the displacement of the talc particles [16].