## 4 Discussion
The exact reason behind the deteriorated performance of the MGD samples remains uncertain, but it is clear that compositional changes take place in both the PL and NPL lipids. Furthermore, dMGS may consist of up to 22 wt % of non-lipid components (i.e., proteins, salts, and polysaccharides) and these in turn might disturb the normal functionality of tear lipids [3; 4; 30]. Therefore, the ability of ophthalmic nanoemulsions to improve the spread and organization of MGS layersin vitro via supplementation of polar and nonpolar lipid molecules serves as a trustworthy indicator of the formulations' potential for use in clinical settings in vivo [10; 31]. As shown in a previous study [12], similarly to other oil-in-water ophthalmic nanoemulsions with comparable compositions, RDA films exhibited typical behavior [24; 25]. Highly reversible, non-collapsible surface films are formed by nanoemulsion. As demonstrated previously, sesame oil can enhance the nonpolar lipids in MGS films and promote the development of a more homogeneous and well-organized NPL stratum [9]. In contrast to the analysis of pure sesame oil, it is important to talk about how the eyedrops' amphiphilic ingredients (CO-10, MYS-40, Poloxamer 407, and menthol) may affect how RDA interacts with MGS films and how they may help the eyedrop interact with the meibum films. According to a recent study [31], surfactants like MYS-40, which have a high hydrophilic-lipophilic balance, can make up for a moderate deficiency of polar lipids by incorporating into the TFLL at the interface with the aqueous tear. Consequently, it improves the pseudo-binary MGS/RDA films' oil-phase spreading and distribution, as well as the non-polar lipid stratum of the films as a whole. The formulation is optimally miscible with the TFLL when high and low HLB surfactants (CO-10 and MYS-40, respectively) are combined [13; 29]. Poloxamer 407 has been observed to incorporate into lipid films; however, because of its hydrophilic polyethylene oxide moiety, it displays a strong attraction to water and is easily squeezed out in PBS subphase at \(\pi\leq 20\) mN/m in controlled experiments involving both MGS layers and phospholipid monolayers [32]. It has been discovered that menthol can enhance the PL-coated MGS/aqueous interface through its interaction with lipid films. The spread of MGS layers and their dilatational viscoelasticity are improved by the interaction. The outcomes are consistent with recent studies that show that patients with dry eye syndrome had their tear films become more stable after using warm compresses infused with menthol [33]. Earlier studies showed that RDA and its constituents exert a number of potentially clinically beneficial effects on \"healthy\" tear lipids in vitro: (i) sesame oil locates on top of the thinner (darker as observed by BAM) monolayer patches in the SO/nMGS layers and increases the overall uniformity and thickness of \"healthy\" meibum films [9]; and (ii) supplementation with RDA accelerates and stabilizes the spread of nMGS at the model tear surface, as seen by the displacement of talcum particles used as visualizer [12]. It is indicative that the latter outcome cannot be accomplished by the supplementation of an extra amount of nMGS to the layers. This observation is consistent with the classical finding of [PERSON] and [PERSON] [34] that MGS functions as a \"tracer oil\" with limited and nonuniform spread, and that it may require either tear-intrinsic or exogenous (i.e., supplemented via eyedrops) surfactants to improve the performance of Meibomian oil at the air/tear interface. In this study, the challenge to RDA was extended, as here the RDA capability was tested as to its ability to restore the functionality of \"diseased\" meibum, dMGS, collected from MGD patients.