Another Brick in the Wall of Tear Film Insights Added Through the Total Synthesis and Biophysical Profiling of anteiso-Branched Wax and Cholesteryl Esters.

The tear film lipid layer (TFLL) plays a vital part in maintenance of ocular health and represents a unique biological barrier comprising unusual and specialized lipid classes and species. The wax and cholesteryl esters (WEs and CEs) constitute roughly 80-90% of the TFLL. The majority of species in these lipid classes are branched and it is therefore surprising that the synthesis and properties of the second largest category of species, i.e., the anteiso-branched species, remain poorly characterized. In this study, we have developed a total synthesis route and completed a detailed NMR spectroscopic characterization of two common anteiso-branched species, namely: (22S)-22-methyltetracosanyl oleate and cholesteryl (22'S)-22'-methyltetracosanoate. In addition, we have studied their structural properties in the bulk state by wide-angle and small-angle X-ray scattering and their behavior at the aqueous interface using Langmuir monolayer techniques. A comparison to the properties displayed by iso-branched and straight-chain analogues indicate that branching patterns lead to distinct properties in the CE and WE lipid classes. Overall, this study complements the previous work in the field and adds another important brick in the tear film insights wall.

New Insights into the Molecular Structure of Tear Film Lipids Revealed by Surface X-ray Scattering.

The tear film lipid layer (TFLL) is a unique biological membrane that serves a pivotal role in the maintenance of ocular surface health. Reaching an overarching understanding of the functional principle of the TFLL has been hampered by a lack of insights into the structural and functional roles played by individual lipid classes. To bridge this knowledge gap, we herein focus on studying films formed by principal lipid classes by surface scattering methods. Through grazing incidence X-ray diffraction and X-ray reflectivity studies, we reveal quantitative data about the lattice distances, molecular tilt angles, and mono/multilayer thickness and density profiles for central TFLL lipid classes under close to simulated physiological conditions. In addition, we discuss the correlation of the results to those obtained previously with the natural lipid composition of meibum.

Early-Stage Development of an Anti-Evaporative Liposomal Formulation for the Potential Treatment of Dry Eyes.

Dry eye disease (DED), the most common ocular disorder, reduces the quality of life for hundreds of millions of people annually. In healthy eyes, the tear film lipid layer (TFLL) stabilizes the tear film and moderates the evaporation rate of tear fluid. In >80% of DED cases, these central features are compromised leading to tear film instability and excessive evaporation of tear fluid. Herein we assess the potential of liposomal formulations featuring phosphatidylcholines and tailored lipid species from the wax ester and O-acyl-ω-hydroxy fatty acid categories in targeting this defect. The developed lead formulation displays good evaporation-resistant properties and respreadability over compression-expansion cycles in our Langmuir model system and a promising safety and efficacy profile in vitro. Preclinical in vivo studies will in the future be required to further assess and validate the potential of this concept in the treatment of DED.

Biophysical profiling of synthetic ultra-long tear film lipids.

The tear film lipid layer (TFLL) is a unique biological membrane of importance to the maintenance of ocular surface health. The underlying factors at play, e.g. the ability to retard evaporation and offer protection from the environment, are all closely connected to the properties of individual lipid components and their interplay. The TFLL contains unique ultra-long polar lipid species such as O-acyl-ω-hydroxy fatty acids, type I-St diesters and type II diesters, which are considered important for its proper function. Herein, we have synthesized model compounds from these categories and studied their biophysical and surface rheological properties at the aqueous interface. Altogether, we provide insights on the distinct biophysical profiles of these lipid classes and discuss how their interplay may affect the structure and function of the TFLL.

On the importance of chain branching in tear film lipid layer wax and cholesteryl esters.

The tear film lipid layer (TFLL) is important to the maintenance of ocular surface health. Surprisingly, information on the individual roles of the myriad of unique lipids found therein is limited. The most abundant lipid species are the wax esters (WE) and cholesteryl esters (CE), and, especially their branched analogs. The isolation of these lipid species from the TFLL has proved to be tedious, and as a result, insights on their biophysical profiles and role in the TFLL is currently lacking. Herein, we circumvent these issues by a total synthesis of the most abundant iso-methyl branched WEs and CEs found in the TFLL. Through a detailed characterization of the biophysical properties, by the use of Langmuir monolayer and wide-angle X-ray scattering techniques, we demonstrate that chain branching alters the behavior of these lipid species on multiple levels. Taken together, our results fill an important knowledge gap concerning the structure and function of the TFLL on the whole.

Tear Film Lipid Layer Structure: Self-Assembly of O-Acyl-ω-hydroxy Fatty Acids and Wax Esters into Evaporation-Resistant Monolayers.

In healthy eyes, the tear film lipid layer (TFLL) is considered to act as an evaporation resistant barrier, which prevents eyes from drying. Seeking to understand the mechanisms behind the evaporation resistance of the TFLL, we studied mixtures of lipid layer wax esters and O-acyl-ω-hydroxy fatty acids. Analyzing their self-assembly and biophysical properties led to new discoveries concerning the structure and function of the TFLL. We discovered how these lipids self-assemble at the air-water interface and form an efficient antievaporative barrier, demonstrating for the first time how the interaction of different tear film lipid species can improve the evaporation resistance compared with individual lipid classes on their own. These results provide a potential mechanism for the evaporation resistance of the lipid layer. In addition, the results serve as a base for the future development of improved dry eye treatments and other applications where the evaporation of water represents a significant challenge.

The Properties and Role of O-Acyl-ω-hydroxy Fatty Acids and Type I-St and Type II Diesters in the Tear Film Lipid Layer Revealed by a Combined Chemistry and Biophysics Approach.

The tear film lipid layer (TFLL) that covers the ocular surface contains several unique lipid classes, including O-acyl-ω-hydroxy fatty acids, type I-St diesters, and type II diesters. While the TFLL represents a unique biological barrier that plays a central role in stabilizing the entire tear film, little is known about the properties and roles of individual lipid species. This is because their isolation from tear samples in sufficient quantities is a tedious task. To provide access to these species in their pure form, and to shed light on their properties, we here report a general strategy for the synthesis and structural characterization of these lipid classes. In addition, we study the organization and behavior of the lipids at the air-tear interface. Through these studies, new insights on the relationship between structural features, such as number of double bonds and the chain length, and film properties, such as spreading and evaporation resistance, were uncovered.

Interactions of polar lipids with cholesteryl ester multilayers elucidate tear film lipid layer structure.

PURPOSE: The tear film lipid layer (TFLL) covers the tear film, stabilizing it and providing a protective barrier against the environment. The TFLL is divided into polar and non-polar sublayers, but the interplay between lipid classes in these sublayers and the structure-function relationship of the TFLL remains poorly characterized. This study aims to provide insight into TFLL function by elucidating the interactions between polar and non-polar TFLL lipids at the molecular level. METHODS: Mixed films of polar O-acyl-ω-hydroxy fatty acids (OAHFA) or phospholipids and non-polar cholesteryl esters (CE) were used as a model of the TFLL. The organization of the films was studied by using a combination of Brewster angle and fluorescence microscopy in a Langmuir trough system. In addition, the evaporation resistance of the lipid films was evaluated. RESULTS: Phospholipids and OAHFAs induced the formation of a stable multilamellar CE film. The formation of this film was driven by the interdigitation of acyl chains between the monolayer of polar lipids and the CE multilayer lamellae. Surprisingly, the multilayer structure was destabilized by both low and high concentrations of polar lipids. In addition, the CE multilayer was no more effective in resisting the evaporation of water than a polar lipid monolayer. CONCLUSIONS: Formation of multilamellar films by major tear film lipids suggest that the TFLL may have a similar structure. Moreover, in contrast to the current understanding, polar TFLL lipids may not mainly act by stabilizing the non-polar TFLL sublayer, but through a direct evaporation resistant effect.