## 2 Materials and Methods  
Surface pressure/area (\(\pi\)-A) isotherms were recorded [9] via the Wilhelmy wire probe method (instrumental accuracy 0.01 mN/m) by \(\mu\)Trough XS Langmuir surface balance, area 135 cm\({}^{2}\), volume 100 mL (Kibron, Helsinki, Finland). As a trough subphase, phosphate-buffered saline (PBS, pH 7.4) was used. Meibomian samples were collected by the soft squeeze method [3; 4] 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 documents. The nMGS was provided from five healthy volunteers (4 males, 29.5 \(\pm\) 3.1 years old; 1 female, 54 years old), while dMGS was collected from four MGD patients (2 males, 59 and 79 years old; 2 females, 86 and 90 years old). The difference in age between the sets of sample donors is related to the prevalence profile of dry eye disease; i.e., MGD and dry eye in general prevail in the population of elderly persons, while optimum tear film stability and lack of signs of dry eye and ocular discomfort is typical for the younger (typically less than fifty years old) age groups [1; 2; 3; 4; 7; 8]. Until its use in experiments, the MGS solutions (1 mg MGS/mL CHCl\({}_{3}\)) were stored at \(-80\)\({}^{\circ}\)C. A mean molecular weight of 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; 13].  
Rohto Dry Aid ophthalmic nanoemulsion (Table 1) (Rohto Pharmaceutical, Osaka, Japan) was used to supplement the dMGS in pseudobinary films [12].  
\begin{table}
\begin{tabular}{c c} \hline \hline
**Active Ingredients 1 ** & **Oil Phase ** (9.12 \(\times\) 10\({}^{-3}\) M)** \\ \hline \multirow{2}{*}{Povidone (3.96 \(\times\) 10\({}^{-5}\) M)} & Sesame Oil/Menthol/Polyoxyethylene 10 \\  & Castor Oil/Polyoxyl 40 Stearate/Poloxamer \\  & 407 = 1.15/0.12/3.2/4.6/0.04 \\ \hline \hline \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 of the components are dissolved in a pH 7.4 saline solution.  
To prevent dust contamination and PBS subphase evaporation, an acrylic cover was installed over the apparatus. Under the cover, relative humidity was kept at over 90% via evaporation of water from open vessels fitted into the acrylic cover chamber. Human MGS was evenly deposited onto the air/water surface using a Hamilton microsyringe. After fifteen minutes were allowed to elapse for the evaporation of chloroform, two symmetrically moving barriers began the dynamic compression-expansion isocycling of the film area at the barrier's maximum rate (70 mm/min), at which no film leakage occurs. Following the third isocycle, the \(\pi\)(A) curves took on a constant form, and those \"stationary\" \(\pi\)(A) isotherms were used for further analysis. All experiments were repeated in triplicate, and there was a less than 2% difference between the isotherms, which ensures the reproducibility and the reliability of the results. The studies were carried out at the physiological temperature of the ocular surface, 35 \({}^{\circ}\)C. A Brewster angle microscope (MC-BAM, Imperx, Boca Raton, FL, USA) was used to observe the films' morphologies.  
To probe the layer's dilatational viscoelasticity a small compression step, \(\Delta\)A/Ao = 5% \(\pm\) 1% (where Ao--initial film area, \(\Delta\)A--area change) was applied to the film equilibrated at initial surface pressure, \(\pi_{0}\) = 10 mN/m [14; 15; 16; 17; 18; 19]. The observed \(\Delta\)\(\pi\) relaxation transients were analyzed further.