Meibum Films Research Articles

Surface Chemistry Study of Normal and Diseased Human Meibum Films Prior to and after Supplementation with Tear Mimetic Eyedrop Formulation

Ophthalmic nanoemulsions that can treat the deficiencies of meibum (MGS) in Meibomian gland disease and restore its functionality in the tear film are greatly sought. The Rohto Dry Aid (RDA) formulation employs TEARSHIELD TECHNOLOGYTM, which uses a multicomponent oil phase of polar and non-polar lipid-like molecules selected to mimic the profiles of healthy meibum. Thus, the interactions of RDA with “diseased” Meibomian (dMGS) films merit deeper analysis, as these interactions might offer important clues for both the development of new ocular formulations and the processes behind the therapeutic action of the nanoemulsions. Pseudobinary dMGS/RDA films were spread at the air–water surface of the Langmuir trough. Surface pressure-area isocycles and stress relaxations were used to access the layer’s response to blink-like cycling and dilatational viscoelasticity, respectively, while film morphology was recorded via Brewster angle microscopy. It was found that RDA is able to reverse the brittleness and to restore the stability of “diseased” MGS films and thus to revert the layer’s properties to the functionality of healthy Meibomian lipids. Therefore, in order to effectively treat dry eyes with MGS-oriented therapy, ophthalmic nanoemulsions warrant more research.

Langmuir Trough Study of the Interactions of Tear Mimetic Eyedrop Formulation with Human Meibum Films

Meibomian gland disease is associated with quantitative or qualitative deficiencies of meibum (MGS) that result in tear film instability. Thus, there is great demand for ophthalmic nanoemulsions that can replenish MGS and recover its performance at the air/tear surface. Rohto Dry Aid (RDA) utilizes TEARSHIELD TECHNOLOGYTM implementing a complex oil phase of non-polar and polar lipid-like molecules. Therefore, the interactions of RDA with MGS surface films deserve further study as they may provide valuable insights (i) into the mechanisms behind the nanoemulsion therapeutic action and (ii) for the design of novel ophthalmic formulations. Pseudobinary meibum/RDA films were formed at the air–water surface of the Langmuir trough. Surface pressure-area isocycles and stress relaxations were employed to probe the layer (i) reorganization upon cycling and (ii) dilatational elasticity, respectively. Film morphology was accessed by Brewster angle microscopy and the spreading properties of RDA-supplemented meibum were also probed. The diverse ingredients of the nanoemulsion oil phase complemented the non-polar and polar lipid constituents of the meibomian layers, which resulted in enhanced continuity of the MGS duplex film structure and facilitated the MGS spread and viscoelasticity. Nanoemulsions deserve further study as a potent tool for MGS-oriented therapy for dry eyes.

Correlations between bulk and surface properties of meibomian lipids with alteration of wax-to-sterol esters content

In a recent study (Ewurum et al., 2021), wax (WE) and sterol esters (CE) from human meibum secretions (MGS) were separated and reconstituted with controlled WE/CE ratios (0%, 20%, 30%, 40%, 50%, 75% and 100% CE weight fractions). It was found that the alterations in the CE content of WE/CE mixtures modified the hydrocarbon chain conformation and packing of the mixture. A major question that emerges is whether the spectroscopic packing parameters determined for bulk meibum translate to a change in the performance of meibomian layers at the air/water interface, as it is the surface film functionality that is crucial for the performance of MGS at the ocular surface. The study of human meibum films with Langmuir surface balance was performed to access the surface properties at blink-like deformations of the film area. Surface pressure (π)-area (A) isocycles and stress relaxations were used to assess the layer’s reorganization during area cycling and dilatational elasticity, respectively. The morphology of the films was monitored by Brewster angle microscopy. It was found that the increased order and chain melting temperature of the bulk samples correlated with a raise in the maximum surface pressure attained at minimal surface area and in the transient dilatational modulus of the meibomian layers. Such correlations may allow for development of an improved understanding between the bulk and surface properties of human meibum and of other natural and synthetic tear lipid films.

Interactions of Meibum and Tears with Mucomimetic Polymers: A Hint towards the Interplay between the Layers of the Tear Film.

Recent clinical findings suggest that mucomimetic polymers (MMP) can alter not only the texture of the aqueous tear but also the spreading and structure of the tear film (TF) lipid layer, thereby allowing for their synchronized performance in vivo. Thus, we aimed to evaluate in vitro (i) the capability of pharmaceutically applicable MMP to ensure the formation of post-evaporative ferning patterns (a characteristic feature of the “healthy” tear colloid) and (ii) the MMP interactions with human meibum films accessed in the course of blink-like deformations via Langmuir surface balance and Brewster angle microscopy (BAM). Four MMP were used- hyaluronic acid (HA), cross-linked hyaluronic acid (CHA), carboxymethyl cellulose (CMC) and gellan gum (GG)- at the concentrations of 0.0001%, 0.001%, 0.01%, 0.05% and 0.1%. Significant differences were observed in the MMP fern formation capability: CHA (≥0.001%) > HA (≥0.01%) = CMC (≥0.01%) > GG (≥0.05%). All MMP affected the spreading of meibum, with BAM micrographs revealing thickening of the films. CHA was particularly efficient, showing concentration-dependent enhancement of tear ferning and of meibomian layer structure, surfactant properties and viscoelasticity. Thus, endogenous and exogenous MMP may play key roles for the concerted action of the TF layers at the ocular surface, revealing novel routes for TF-oriented therapeutic applications.

Evaporation retardation by model tear-lipid films: The roles of film aging, compositions and interfacial rheological properties

A novel methodology for assessing evaporation (up to 48 h) through lipid-nanofilms in vitro was developed. The influence of lipid-mixture compositions on evaporation rates was studied. The evaporative fluxes and rheology of lipid-nanofilms were compared with those of human tear-lipid nanofilms in vitro.A sessile-drop technique with precise drop-volume control was adapted to measure evaporation rates at constant temperature of 36 °C and humidity of 75 %. Model lipid solutions were deposited on the surface of aqueous drops to create nanofilms of 10–100 nm. The measurements of dynamic surface pressure vs. nanofilm-thickness were performed under the same conditions. The lipid-mixtures compositions were chosen to mimic that of human tear lipids. Evaporation through lipid nanofilms decreased with film thickness and aging. Evaporation through 70-nm films was 2.5–3 time slower than through 10-nm-thick films. Nonpolar-lipid mixtures reduced evaporation by approximately 35 %. The optimized model-lipid mixtures containing polar phospholipids reduced evaporation by 70–75 %, matching the evaporation-reduction by human-lipid nanofilms in vitro. These model mixtures exhibited interfacial rheology similar to human tear lipids in vitro.This methodology substantiated that aged lipid-nanofilms significantly reduced evaporation in vitro. These findings contradict to the previous reports suggesting that model lipid and meibum films do not retard evaporation in vitro. Polar phospholipids enhance evaporative resistance close to the level observed for human tear-lipid films in vivo. We hypothesize that unique rheological properties of tear-lipid nanofilms are germane to the specific mono- and bi-layered structures formed by phospholipids at the lipid-air and lipid-aqueous interfaces.

Insights into Tear Film Stability from Babies and Young Adults: A Study of Human Meibum Lipid Conformation and Rheology.

Babies have the most stable tears and people with dry eye have the least stable tears. Meibum may contribute to tear film stability, so in this study, the hydrocarbon chain conformation and rheology of meibum from babies was studied for the first time. Infrared spectroscopy was used to measure lipid phase transitions. Rheology was measured using Langmuir film technology. Meibum from 25 donors 1 to 13 years old was compared with meibum from 18 donors 13 to 25 years old. The phase transition temperature and lipid order (stiffness) increased with increasing age from 1 to 25 years. The increase in meibum lipid order from 1 to 25 years of age may contribute to the instability of the tear film with age and contribute to films with a higher reciprocal compressibility modulus that are not as compressible and not as viscoelastic. Changes in the lipid phase transition parameters of meibum lipid with dry eye are an exacerbation of the changes observed with age. The lower reciprocal compressibility moduli of meibum films from children and babies compared with meibum from adults reiterates higher stability in their films which spread better, resist deformation, and facilitates their ability to be quickly restored after blinking.

Effects of Lipid Saturation on the Surface Properties of Human Meibum Films.

Elevated levels of acyl chain saturation of meibomian lipids are associated with vastly different effects: from enhanced tear film (TF) stability in infants to shortened TF breakup time in meibomian gland disease patients. Thus it is important to study the effect of saturation on the surface properties of human meibum (MGS). Therefore, MGS films (1, 2, 3, 4, 5, 10, 25, 50, 67, and 100% saturation) were spread at the air/water interface of a Langmuir surface balance. The layers’ capability to reorganize during dynamic area changes was accessed via the surface pressure (π)-area (A) compression isotherms and step/relaxation dilatational rheology studies. Film structure was monitored with Brewster angle microscopy. The raise in the % (at ≥10%) of saturation resulted in the formation of stiffer, thicker, and more elastic films at π ≥ 12 mN/m with the effects being proportional to the saturation level. At the same time, at low (≤10 mN/m) π the raise in saturation resulted in altered spreading and heterogeneous structure of MGS layers. The strong impact of saturation on MGS surface properties correlates with our recent spectroscopy study, which demonstrated that saturation induced increase of MGS acyl chain order, phase transition temperature, and cooperativity.

Surface chemistry of the interactions of cationic nanoemulsions with human meibum films

PurposeIkervis® (IKV) cationic nanoemulsions (CNE) were demonstrated to enhance tear film stability in vivo possibly via effect on tear film lipid layer (TFLL). Therefore the interactions of IKV and of binary and ternary mixtures of its constituents were studied with human meibum (MGS) films. The binary mixtures consisted of 2% mid chain triglycerides (MCT) blended with 0.005% cetalkonium chloride (CKC) and 0.3% Tyloxapol (e.g. MCT/CKC and MCT/Tylo respectively). The ternary mixture contained MCT/CKC/Tylo (2%/0.005%/0.3%). The impact of 0.1 μM bovine submaxillary mucin (BSM) on CNE/MGS interactions was also evaluated.MethodsMGS and CNE oils were spread at the air/water interface of a Langmuir surface balance in range of 2D ratios (20/1, 10/1, 5/1, 3/1, 2/1 and 1/1) at two measurement regimes: with (i) MGS or (ii) total lipid amount kept constant. The films capability to reorganize during dynamic area cycling was evaluated. The layers dilatational rheology was probed via the step/relaxation method. Films structure was monitored with Brewster Angle microscopy.ResultsThe binary mixtures showed limited spreading and miscibility with MGS resulting in poor mechanical properties. The ternary mixture and IKV spread and mixed well with MGS. At fixed MGS amount, the inclusion of CNEs enhanced the structure, properties and elasticity of the layers. At fixed total lipid, the films remained primarily elastic, but at high (≤3/1) CNE content the elasticity slightly decreased and heterogeneities in layers structure were observed. BSM enhanced the ternary mixture (IKV)/ MGS interactions.ConclusionsAt physiologically relevant MGS/CNE ratios MCT/CKC/Tylo and IKV interact favorably with MGS films. The positive effect of BSM suggests that polyanionic polysaccharides can enhance CNE/TFLL interactions in vivo.Support: Collaborative study grant by Santen SAS, Evry, France.

Evaporation and Hydrocarbon Chain Conformation of Surface Lipid Films

PurposeThe inhibition of the rate of evaporation (Revap) by surface lipids is relevant to reservoirs and dry eye. Our aim was to test the idea that lipid surface films inhibit Revap. MethodsRevap were determined gravimetrically. Hydrocarbon chain conformation and structure were measured using a Raman microscope. Six 1-hydroxyl hydrocarbons (11-24 carbons in length) and human meibum were studied. Reflex tears were obtained from a 62-year-old male. ResultsThe Raman scattering intensity of the lipid film deviated by about 7 % for hydroxyl lipids and varied by 21 % for meibum films across the entire film at a resolution of 5 μm2. All of the surface lipids were ordered. Revap of the shorter chain hydroxyl lipids were slightly (7%) but significantly lower compared with the longer chain hydroxyl lipids. Revap of both groups was essentially similar to that of buffer. A hydroxyl lipid film did not influence Revap over an estimated average thickness range of 0.69 to >6.9 μm. Revap of human tears and buffer with and without human meibum (34.4 μm thick) was not significantly different. Revap of human tears was not significantly different from buffer. ConclusionsHuman meibum and hydroxyl lipids, regardless of their fluidity, chain length, or thickness did not inhibit Revap of buffer or tears even though they completely covered the surface. It is unlikely that hydroxyl lipids can be used to inhibit Revap of reservoirs. Our data do not support the widely accepted (yet unconfirmed) idea that the tear film lipid layer inhibits Revap of tears.

Surface properties and exponential stress relaxations of mammalian meibum films

The surface properties of meibomian secretion (MGS), the major constituent of the tear film (TF) lipid layer, are of key importance for TF stability. The interfacial properties of canine, cMGS, and feline, fMGS, meibum films were studied using a Langmuir surface balance. These species were selected because they have blinking frequency and TF stability similar to those of humans. The sample's performance during dynamic area changes was evaluated by surface pressure (π)-area (A) isocycles and the layer structure was monitored with Brewster angle microscopy. The films' dilatational rheology was probed via the stress-relaxation technique. The animal MGS showed similar behavior both between each other and with human MGS (studied previously). They form reversible, non-collapsible, multilayer thick films. The relaxations of canine, feline, and human MGS films were well described by double exponential decay reflecting the presence of two processes: (1) fast elastic process, with characteristic time τ<10s and (2) slow viscous process, with τ>100s-emphasizing the meibum layers viscoelasticity. The temperature decrease from 35 to 25°C resulted in decreased thickness and lateral expansion of all MGS layers accompanied with increase of the π/A hysteresis and of the elastic process contribution to π relaxation transients. Thus, MGS films of mammals with similar blinking frequency and TF stability have similar surface properties and stress relaxations unaltered by the interspecies MGS compositional variations. Such knowledge may impact the selection of animal mimics of human MGS and on a better understanding of lipid classes' impact on meibum functionality.

Sebum/Meibum Surface Film Interactions and Phase Transitional Differences.

PurposeSebum may contribute to the composition of the tear film lipid layer naturally or as a contaminant artifact from collection. The aims of this study were to determine: if sebum changes the rheology of meibum surface films; if the resonance near 5.2 ppm in the 1H-NMR spectra of sebum is due to squalene (SQ); and if sebum or SQ, a major component of sebum, interacts with human meibum.MethodsHuman meibum was collected from the lid margin with a platinum spatula. Human sebum was collected using lipid absorbent tape. Langmuir trough technology was used to measure the rheology of surface films. Infrared spectroscopy was used to measure lipid conformation and phase transitions. We used 1H-NMR to measure composition and confirm the primary structure of SQ.ResultsThe NMR resonance near 5.2 ppm in the spectra of human sebum was from SQ which composed 28 mole percent of sebum. Both sebum and SQ lowered the lipid order of meibum. Sebum expanded meibum films at lower concentrations and condensed meibum films at higher concentrations. Sebum caused meibum to be more stable at higher pressures (greater maximum surface pressure).ConclusionsPhysiological levels of sebum would be expected to expand or fluidize meibum making it spread better and be more surface active (qualities beneficial for tear film stability). Sebum would also be expected to stabilize the tear film lipid layer, which may allow it to withstand the high shear pressure of a blink.

Lipid order, saturation and surface property relationships: A study of human meibum saturation

Tear film stability decreases with age however the cause(s) of the instability are speculative. Perhaps the more saturated meibum from infants may contribute to tear film stability. The meibum lipid phase transition temperature and lipid hydrocarbon chain order at physiological temperature (33 °C) decrease with increasing age. It is reasonable that stronger lipid–lipid interactions could stabilize the tear film since these interactions must be broken for tear break up to occur. In this study, meibum from a pool of adult donors was saturated catalytically. The influence of saturation on meibum hydrocarbon chain order was determined by infrared spectroscopy. Meibum is in an anhydrous state in the meibomian glands and on the surface of the eyelid. The influence of saturation on the surface properties of meibum was determined using Langmuir trough technology. Saturation of native human meibum did not change the minimum or maximum values of hydrocarbon chain order so at temperatures far above or below the phase transition of human meibum, saturation does not play a role in ordering or disordering the lipid hydrocarbon chains. Saturation did increase the phase transition temperature in human meibum by over 20 °C, a relatively high amount. Surface pressure–area studies showing the late take off and higher maximum surface pressure of saturated meibum compared to native meibum suggest that the saturated meibum film is quite molecularly ordered (stiff molecular arrangement) and elastic (molecules are able to rearrange during compression and expansion) compared with native meibum films which are more fluid agreeing with the infrared spectroscopic results of this study. In saturated meibum, the formation of compacted ordered islands of lipids above the surfactant layer would be expected to decrease the rate of evaporation compared to fluid and more loosely packed native meibum. Higher surface pressure observed with films of saturated meibum compared to native meibum suggests greater film stability especially under the high shear stress of a blink.

Surface chemistry study of the interactions of hyaluronic acid and benzalkonium chloride with meibomian and corneal cell lipids

Hyaluronic acid (HA) is a non-surface active water soluble anionic polyelectrolyte found in sub-nanomolar levels in human tears and implemented in substitutes of human tears' aqueous layer. In the current work we studied the interactions of high molecular weight (Mw 1 × 106 Da) HA with films of ocular surface lipids and investigated the potential of HA to modify the interactions of the lipid films with the low Mw cationic surfactant dodecyldimethylbenzylammonium chloride (C12-BAC) known for its adverse effects on tear film compounds. The interactions of HA and/or C12-BAC with meibomian and corneal lipid films were examined using a Langmuir surface balance. The film morphology was monitored by Brewster Angle Microscopy. The HA/C12-BAC interactions at the air/water interface and in the solution bulk were also examined. HA interacted with meibomian lipids and enhanced the structure and the rheological properties of the meibum films. HA bound C12-BAC in bulky polymer/surfactant complexes and at ≥0.1% HA neutralized the capability of C12-BAC to penetrate the lipid layers and to disrupt their integrity and viscoelasticity. The results were correlated with the capability of HA to maintain the viability of C12-BAC treated corneal cells. Our study suggests a possible novel implementation of high Mw anionic polyelectrolytes like HA: the polymers can be instilled on the ocular surface together with cationic surfactant containing drugs, and increase the biocompatibility of the ophthalmic formulations. A molecular scale mechanism is proposed for the interactions of HA with tear lipids and cationic surfactants.

Molecular Structure of Interfacial Human Meibum Films

Meibum is the primary component of the tear film lipid layer. Thought to play a role in tear film stabilization, understanding the physical properties of meibum and how they change with disease will be valuable in identifying dry eye treatment targets. Grazing incidence X-ray diffraction and X-ray reflectivity were applied to meibum films at an air-water interface to identify molecular organization. At room temperature, interfacial meibum films formed two coexisting scattering phases with rectangular lattices and next-nearest neighbor tilts, similar to the Ov phase previously identified in fatty acids. The intensity of the diffraction peaks increased with compression, although the lattice spacing and molecular tilt angle remained constant. Reflectivity measurements at surface pressures of 18 mN/m and above revealed multilayers with d-spacings of 50 Å, suggesting that vertical organization rather than lateral was predominantly affected by meibum-film compression.

Surface Chemistry Study of the Interactions of Pharmaceutical Ingredients with Human Meibum Films

To perform a surface chemistry study of the interactions between the benzalkonium chloride (BAC)-preserved eyedrops Travatan, the SofZia-preserved TravatanZ, and the Polyquad-preserved DuoTrav, and tear film (TF) constituents. The interactions of TF compounds with the individual preservatives, BAC, SofZia, and Polyquad, were also examined. Langmuir surface balance measurements were used to examine the interactions between the pharmaceuticals and films of human meibum and rabbit corneal cell lipid extracts. Surface pressure-area isocycles were used to assess the sample's capability to compress and spread during dynamic area changes. The dilatational rheologic properties of human meibum films, pure and in the presence of preservatives, were probed by stress-relaxation studies. Lipid film morphology was monitored by Brewster angle microscopy. The viability of SofZia- and Polyquad-treated Statens Seruminstitut Rabbit Cornea (SIRC) cell cultures was also evaluated. The interactions between BAC-preserved eyedrops and lipids resulted in impaired lipid spread, formation of discontinuous nonuniform surface layers, and increased surface pressure-area hysteresis during compression/expansion. In contrast, TravatanZ, DuoTrav, and the individual preservatives SofZia and Polyquad proved to be safe to the lipid film structure and isothermal reversibility. The stress-relaxation experiments revealed that the viscoelastic properties of meibomian film are impaired by BAC, and remain unaffected by SofZia and Polyquad. SIRC cells' viability and capability to form confluent cellular monolayer were also maintained after exposure to SofZia and Polyquad. Surface chemistry studies present criteria for preclinical in vitro molecular scale characterization of the interactions between eyedrop compounds and TF constituents.

Temperature-Induced Transitions in the Structure and Interfacial Rheology of Human Meibum

Meibomian lipids are the primary component of the lipid layer of the tear film. Composed primarily of a mixture of lipids, meibum exhibits a range of melt temperatures. Compositional changes that occur with disease may alter the temperature at which meibum melts. Here we explore how the mechanical properties and structure of meibum from healthy subjects depend on temperature. Interfacial films of meibum were highly viscoelastic at 17°C, but as the films were heated to 30°C the surface moduli decreased by more than two orders of magnitude. Brewster angle microscopy revealed the presence of micron-scale inhomogeneities in meibum films at higher temperatures. Crystalline structure was probed by small angle x-ray scattering of bulk meibum, which showed evidence of a majority crystalline structure in all samples with lamellar spacing of 49 Å that melted at 34°C. A minority structure was observed in some samples with d-spacing at 110 Å that persisted up to 40°C. The melting of crystalline phases accompanied by a reduction in interfacial viscosity and elasticity has implications in meibum behavior in the tear film. If the melt temperature of meibum was altered significantly from disease-induced compositional changes, the resultant change in viscosity could alter secretion of lipids from meibomian glands, or tear-film stabilization properties of the lipid layer.