Meibomian Lipid Films Research Articles

INTERACTION OF NANOSTRUCTURED LIPID CARRIERS WITH HUMAN MEIBUM

Objective: This study aimed to determine the possibility of nanostructured lipid carriers (NLCs) as a bionic tear film by determining the surface activities of the developed NLCs and their interaction with human meibomian lipid films.
 Methods: NLCs with different types of solid lipids and surfactants were prepared by a high-pressure homogenizer. The particle size was determined by dynamic light scattering. The surface activities of the NLCs and NLCs mixed with meibomian lipids were measured using a Langmuir trough and the resulting surface pressure area (Π-A) profiles were compared. These lipid films were further analyzed using fluorescence microscopy and scanning electron microscopy (SEM).
 Results: The particle size of prepared NLCs varied from 38–280 nm based on types of solid lipid and surfactant. All NLCs were highly surface active as indicated by their maximum surface pressure (Πmax). The Π-A profiles of meibum seeded with NLCs showed higher surface pressure than meibum alone and the shape of profiles were dominated by the meibomian lipids. These findings were in agreement with fluorescence and SEM micrographs, which revealed that the NLCs could adsorb and integrate to the meibomian lipid films as well as diffuse from the subphase to the lipid films.
 Conclusion: NLCs are surface active and can integrate with meibomian lipid films formed stable films. The type of interaction can be tailored by altering the solid lipids used in the formulation of the NLCs which could provide the means to develop efficient formulations for targeting dry eye disease related to a non-functional tear film lipid layer.

Biophysical investigations of the structure and function of the tear fluid lipid layers and the effect of ectoine. Part B: Artificial lipid films

The tear fluid lipid layer is present at the outermost part of the tear film which lines the ocular surface and functions to maintain the corneal surface moist by retarding evaporation. Instability in the structure of the tear fluid lipid layer can cause an increased rate of evaporation and thus dry eye syndrome. Ectoine has been previously shown to fluidize lipid monolayers and alter the phase behavior. In the current study we have investigated the effect of ectoine on the artificial tear fluid lipid layer composed of binary and ternary lipid mixtures of dipalmitoyl phosphatidylcholine (DPPC), cholesteryl esters and tri-acyl-glycerols. The focus of our study was mainly the structural and the biophysical aspects of the artificial tear fluid lipid layer using surface activity studies and topology analysis. The presence of ectoine consistently causes an expansion of the pressure–area isotherm indicating increased intermolecular spacing. The topology studies showed the formation of droplet-like structures due to the addition of ectoine only when tri-acyl-glycerol is present in the mixture of DPPC and chol-palmitate, similar to the natural meibomian lipids. Consequently, the hypothesis of an exclusion of tri/di-acyl-glycerol from the meibomian lipid film in the presence of ectoine in the subphase is confirmed. A model describing the effect of ectoine on meibomian lipid films is further presented which may have an application for the use of ectoines in eye drops as a treatment for the dry eye syndrome.

Biophysical investigations of the structure and function of the tear fluid lipid layer and the effect of ectoine. Part A: Natural meibomian lipid films

The tear fluid lipid layer is the outermost part of the tear film on the ocular surface which protects the eye from inflammations and injuries. We investigated the influence of ectoine on the structural organization of natural meibomian lipid films using surface activity analysis and topographical studies. These films exhibit a continuous pressure–area isotherm without any phase transition. With the addition of ectoine, the isotherm is expanded towards higher area per molecule values suggesting an increased area occupied by the interfacial lipid molecules. The AFM topology scans of natural meibomian lipid films reveal a presence of fiber-like structures. The addition of ectoine causes an appearance of droplet-like structures which are hypothesized to be tri-acyl-glycerols and other hydrophobic components excluded from the lipid film. Further the material properties of the droplet-like structure with respect to the surrounding were determined by using the quantitative imaging mode of the AFM technique. The droplet-like structures were found to be comparatively softer than the surrounding. Based on the observations a preliminary hypothesis is proposed explaining the mechanism of action of ectoine leading to the fluidization of meibomian lipid films. This suggests the possibility of ectoine as a treatment for the dry eye syndrome.

The Effect of Ectoins on the Structural Organization of the Tear Fluid Monolayer

We investigate the influence of Ectoin on the structural organization of the natural and artificial tear fluid lipid layers (ATFLL) using surface activity analysis and topographical studies. The natural meibomian lipids exhibit a continuous pressure-area isotherm without any phase transitions. In the presence of ectoin, the isotherm is expanded towards higher area per molecule implying decreased interaction between the lipid molecules. The AFM scans show presence of fiber like structures in the natural meibomian lipid film. In the presence of ectoine, droplet-like structures are observed which are hypothesized to be triacyl glycerols excluded from the lipid film. ATFLL illustrate the fluidizing effect of ectoine on the lipid films where the pressure-area isotherms are expanded in the presence of ectoin. With the addition of a triacyl glycerol to the mixture of DPPC and Chol-Palmitate, we observed the formation of similar drop-like structures in the presence of ectoine as in the case of natural meibomian lipid films. Consequently, the hypothesis explaining the exclusion of tri/di acyl glycerol from the meibomian lipid film in the presence of ectoine in the subphase is confirmed which lead us to a model describing the fluidizing effect of ectoine on meibomian lipid films.

An investigation of the likely role of (O-acyl) ω-hydroxy fatty acids in meibomian lipid films using (O-oleyl) ω-hydroxy palmitic acid as a model

(O-acyl) ω-hydroxy fatty acids (OAHFAs) are a recently found group of polar lipids in meibum. Since these lipids can potentially serve as a surfactant in the tear film lipid layer, the surface properties of a molecule of this lipid class was investigated and compared with a structurally related wax ester and a fatty acid. (O-oleyl) ω-hydroxy palmitic acid was synthesized and used as the model OAHFA. It was spread either alone or mixed with human meibum on an artificial tear buffer in a Langmuir trough, and pressure-area isocycle profiles were recorded at different temperatures and compared with those of palmityl oleate and oleic acid. These measurements were accompanied by fluorescence microscopy of meibum mixed films during pressure-area isocycles. The pressure area curves indicated that pure films of the model OAHFA are as surface active as oleic acid films, cover a much larger surface area than either palmityl oleate or oleic acid and show a distinct biphasic pressure-area isocycle profile. The OAHFAs appeared to remain on the aqueous surface and show only a minor re-arrangement into multi-layered structures during repetitive pressure area isocycles. All these properties can be explained by OAHFAs binding weakly to the aqueous surface via an ester group and strongly via a carboxyl group. By contrast, the pressure area profiles of palmityl oleate films indicate that they form multi-layers and oleic acid presumably forms micelles and desorbs into the subphase. When mixed with meibum, similar features as for pure films were observed. In addition, meibum-OAHFA films appeared very homogeneous; a feature not seen with other mixtures. In conclusion these data support the notion that the tested OAHFA is a very potent surfactant which is important in spreading and stabilising meibomian lipid films.

Effects of Keratin and Lung Surfactant Proteins on the Surface Activity of Meibomian Lipids

In vitro studies indicate that surface tension and surface viscosity of the tear film lipid layer (TFLL) are governed by interactions between meibomian lipids and proteins from the aqueous layer. The role of minor tear proteins with strong lipophilic properties or those correlated with pathological states is still unknown. The discovery of lung surfactant proteins (SPs) in tears and keratin in normal and abnormal meibomian gland excretions warrants investigation into their effects on the surface activity of meibomian lipid films. Commercial keratin and bovine lung SPs were used in vitro to assess the surface pressure of meibomian lipid films using a Langmuir trough. The pressure-area profiles of meibomian lipid films seeded with SPs (2.5 μL; 0.1 μg) demonstrated hybrid characteristics between meibomian lipid films alone and SPs alone but reached much higher maximum surface pressures (approximately 30 vs. 24 mN/m). Microscopically, the appearance of meibomian lipid films was not altered by SPs. Maximum surface pressure of meibomian films premixed with keratin was much higher than meibum alone. The pressure-area isocycles appeared more like those of meibomian lipids with a low concentration of protein and more like pure keratin films at a high concentration. The data strongly indicate that SPs and keratin likely interact with the TFLL. SPs are likely to act as strong surfactants and to reduce the surface tension of the lipid layer. Excess concentrations of keratin as identified in patients with meibomian gland dysfunction could disrupt the normal structure of the meibomian lipid film.

Interfacial Dilatational Viscoelasticity of Human Meibomian Lipid Films

Purpose/Aim: The meibomian lipid layer is able to withstand the enormous stresses and deformations that occur during blinking due to the combination of its elastic and viscous properties. The purpose of this study was to measure the dilatational viscoelasticity of in vitro meibomian lipid films and compare how these properties differ between room temperature and physiological temperatures. Viscoelasticity was also compared with meibomian lipid films seeded with cholesterol or β-carotene (the levels of these lipid species change in disease states).Materials and methods: Dilatational viscoelasticity (E) was measured using an oscillating pendant drop method. Measurements were carried out on spread films at the air–water interface as a function of frequency (0.1256–12.56 rad/s) at various temperatures between 18–43 °C.Results: Generally, E gradually decreased as the overall temperature was increased. At both 37 and 20 °C, films demonstrated that the elastic modulus (E′) was more dominant than the viscous modulus (E″), indicating films were more solid-like than fluid-like, regardless of temperature. E′ and E″ were also dependant on frequency, indicating some molecular rearrangements of the lipid molecules as films were compressed and expanded. Films seeded with cholesterol or β-carotene showed a modest increase in the moduli.Conclusions: These results are consistent with previous findings which have predicted and indicated that the meibomian lipid layer is a viscoelastic film at the air–liquid interface. These properties are integral to how the tear film lipid layer is able to maintain its structure, and hence integrity of the ocular surface.

Disruption and Destabilization of Meibomian Lipid Films Caused by Increasing Amounts of Ceramides and Cholesterol

We evaluated quantitatively direct effects of ceramide (Cer) and free cholesterol (FC) on meibomian lipid films (MLF) using a Langmuir trough (LT) and a Brewster angle microscope (BAM). Meibum was obtained from healthy volunteers. A series of mixtures of meibum with Cer or FC (mixed MLF) taken in different ratios were tested. Standard rheologic parameters, such as elasticity and hysteresis of MLF, were computed. BAM was used to study the morphology of MLF. Pure MLF were capable of withstanding multiple compression/expansion cycles with little hysteresis observed (1.9 J/G meibum). The films made of either pure CER or pure FC were clearly collapsible, and had much higher rigidity and hysteresis than pure meibum. Adding progressively higher amounts of CER or FC to meibum had a strong impact on the rigidity, stability, and morphology of the mixed MLF: their hysteresis increased many fold compared to pure meibum. A concomitant increase in the rigidity and collapsibility of the mixed MLF was observed. Cer and FC changed the surface properties of mixed MLF in a way that implied their destabilization and/or disruption. One of the mechanisms that might lead to these effects is strong aggregation of meibum lipids with FC or Cer that leads to the formation of smaller particles of meibum surrounded by a thinner layer of FC or Cer. As Cer and FC can be elevated in meibum and the tear film because of certain pathologic processes, or can be of exogenous nature, our results can explain (partially) a less stable tear film in those subjects.

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.

Lipid Component Contributions to the Surface Activity of Meibomian Lipids

Meibomian lipid films have very complex physical properties that enable them to be compressed and expanded without collapsing. These properties can be attributed to the self assembly of the individual components, mainly wax and cholesteryl esters (WE and CE). Here, the surface pressure properties of WEs and CEs films have been compared to evaluate their contributions to meibomian lipid films. Films of different WEs and CEs were spread on a Langmuir trough and their surface pressure area profiles were compared with a particular emphasis on the effects caused by the degree of saturation of the alkyl/alkene chains. Fully saturated WEs and CEs formed unstable films that collapsed upon compression. Very unsaturated waxes and CEs tended to have two distinct phases, one that reflects interaction with the aqueous subphase, while the second appeared to be with the multilayered bulk of the lipid film. With aging of the films, the WEs tended to move off the surface into the bulk. When meibomian lipid films were seeded with large amounts of WEs, only minor changes could be seen unless the WE was very unsaturated. These data are consistent with meibomian lipid films having a surfactant layer with a complex bulk layer external to this. It is speculated that the bulk layer contains thermotropic smectic chiral liquid crystals of CEs that are interacting with the WEs. This structure would tend to prevent collapse of the meibomian lipids onto the ocular surface and be very tolerant of lipophilic contaminants.

Analysis of Comparison of Human Meibomian Lipid Films and Mixtures with Cholesteryl Esters In Vitro Films using High Resolution Color Microscopy

The lipid layer of the tears has been studied in vivo using high resolution color microscopy (HRCM). The purpose of these experiments was to gain further insight into the structure of the lipid layer by applying HRCM to in vitro meibomian lipid films. Films of human meibomian lipids, cholesteryl nervonate, cholesteryl palmitate, or their mixtures, were spread on a Langmuir trough. Changes to the films were monitored using HRCM as the films were compressed to different surface pressures. The penetration of albumin into a meibomian lipid film also was studied. Small amounts of meibomian lipids at low pressures formed very thin films estimated to be 5.2 nm thick. Compression caused spots to appear in the films. At higher concentrations, micro lenses were a feature of the film. Cholesteryl nervonate formed a multilayered oil slick that did not change with surface pressure. Cholesteryl palmitate formed a stiff film that collapsed at high compression. Mixtures of cholesteryl nervonate and meibomian lipids showed that they mixed to increase surface pressures above that of the individual components. HRCM also allowed albumin to be seen penetrating the meibomian lipid film. HRCM combined with in vitro surface pressure measurements using a Langmuir trough is useful for modeling meibomian lipid films. The films often resemble the appearance of the lipid layer of in vivo films. The data indicate that the lipid layer might be modeled best as a duplex film containing an array of liquid crystals.

Effects of free fatty acids on meibomian lipid films

The purpose of this study was to evaluate the impact of free fatty acids (FFA), namely oleic (OA) and linoleic (LA) ones, on meibomian lipid films (MLF) using a Langmuir trough (LT) and a Brewster angle microscope (BAM). Human meibum was collected from healthy volunteers. A Tris-buffered saline (TBS, pH 7.4) was used as the control aqueous subphase for LT experiments. Then, varying amounts of OA and LA were dissolved in TBS to make FFA-containing subphases. Predetermined amounts of meibum were loaded onto the surface of the (TBS/±FFA) subphases to form MLF. Then, surface pressure-area ( π/A) isotherms of MLF were recorded. Standard rheological parameters such as rigidity, elasticity, and hysteresis, were computed. In a separate experiment, OA and LA were pre-mixed with meibum at different weight ratios prior their spreading onto the control TBS subphase, and the ( π/A) isotherms of the resulting mixed films of meibum and FFA were studied and analyzed in the same fashion as described above. When studied at the normal corneal temperature of 34 °C with the (TBS/−FFA) subphase, meibum formed stable films. When (TBS/+FFA) subphase was used, both FFA quickly disrupted the MLF, acting in a similar fashion. BAM revealed that the most dramatic changes in the structure of MLF occurred in the range of OA concentrations between 5 and 15 μM. However, this effect was apparent even with 2.5 μM OA. When OA was pre-mixed with meibum, but was absent from the subphase, it caused gradual concentration-dependent changes in the ( π/A) isotherms, but the MLF did not disappear from the surface. Thus, tested FFA showed a remarkable ability to disrupt, and/or prevent the formation of, human MLF, which could contribute to the onset of those forms of dry eye disease that are associated with enhanced activity of lipolytic enzymes, such as chronic blepharitis.

Meibomian Lipid Films and the Impact of Temperature

There is evidence that, in cold conditions, the temperature of human eyelids and of the ocular surface drops well below normal physiological levels. This may have a detrimental impact on the stability and functionality of the human tear film and the tear film lipid layer. The goal of this project was to quantitatively examine the possible impact of temperature on the latter. Meibum samples were collected by using a soft-squeezing technique and were studied in a Langmuir trough. The obtained surface pressure and area isotherms were analyzed to determine the biophysical parameters of thin meibomian lipid film (MLF): the lift-off area, collapse pressure, two-dimensional elasticity, and hysteresis and their dependence on temperature. MLF was found to be highly susceptible to changes in temperature. At temperatures below the physiological level, the MLF became stiff and shrank considerably. The shrinkage left a large portion of the air-water interface uncovered with lipid molecules. The effect was shown to be reversible. On reheating, the lipids melted and respread to restore the original film. There was a fundamental difference observed between three-dimensional melting of dry meibum in bulk and the two-dimensional melting in MLF at the air-water interface. Bulk meibum melted in a narrower temperature range and showed a much higher cooperativity of melting. Temperature critically influences MLF. Low temperature leads to stiffening of the film, which loses its ability to form continuous layers at the air-water interface. These effects were shown be of a cooperative nature, manifesting in relatively narrow concentration and temperature ranges.

Adsorption of Human Tear Lipocalin to Human Meibomian Lipid Films

Tear lipocalin (Tlc) is a major lipid binding protein in tears and is thought to have an important role in stabilizing the Meibomian lipid layer by transferring lipids to it from the aqueous layer or ocular surface, or by adsorbing to it directly. These possible roles have been investigated in vitro using human Tlc. Tlc was purified from human tears by size exclusion chromatography followed by ion exchange chromatography. Three additional samples of the Tlc were prepared by lipidation, delipidation, and relipidation. The lipids extracted from the purified Tlc were analyzed by HPLC-MS followed by fragmentation. Adsorption of these different forms of Tlc to a human Meibomian lipid film spread on the surface of an artificial tear buffer in a Langmuir trough were observed by recording changes in the pressure with time (Pi-T profile) and monitoring the appearance of the film microscopically. These results were compared with similar experiments using a bovine Meibomian lipid film. The results indicated that Tlc binds slowly to a human Meibomian lipid film compared with lysozyme or lactoferrin, even at 37 degrees C. The adsorption of Tlc to a human Meibomian lipid film was very different from its adsorption to a bovine Meibomian lipid film, indicating the nature of the lipids in the film is critical to the adsorption process. Similarly, the different forms of Tlc had quite distinct adsorption patterns, as indicated both by changes in Pi-T profiles and the microscopic appearance of the films. It was concluded that human Tlc was capable of adsorbing to and penetrating into a Meibomian lipid layer, but this process is very complex and depends on both the types of lipids bound to Tlc and the lipid complement comprising the Meibomian lipid film.

Adsorption of apo- and holo-tear lipocalin to a bovine Meibomian lipid film

Adsorption of apo- and holo-tear lipocalin (Tlc) to bovine Meibomian lipid film was studied. A Langmuir trough was used for these studies and the adsorption of protein was observed by recording changes in the pressure with time (π–T profile). The films were photographed at different stages of adsorption by doping Meibomian lipids with a fluorescently tagged lipid. The results indicated that apo-Tlc adsorbed much more quickly than holo-Tlc to the Meibomian lipid film. Contrary to the expectation that holo-Tlc would release lipids to the surface and surface pressure would be higher, it was found that the surface pressure was higher with the adsorption of apo-Tlc to the surface. Photography of the films showed that apo- and holo-Tlc interacted differently with the Meibomian lipid layer. Adsorption of holo-Tlc resulted in big bright patches and adsorption of apo-Tlc resulted in many small patches along with the big patches. Both forms of Tlc produced a more stable film as indicated by decreased movement of the protein adsorbed films, and a higher maximum surface pressure upon compression of these films compared with Meibomian lipid films alone. Isocyles of apo-Tlc adsorbed films gave a higher surface pressure than that of holo-Tlc. From these results, it is concluded that both apo- and holo-Tlc adsorbed to the Meibomian lipid layer and the delivery of the lipids from Tlc to the outer lipid layer could not be detected by our techniques. Its scavenging role to remove lipids from the corneal surface and bind with them might be beneficial for increasing tear viscosity but whether those lipids are delivered to the outermost lipid layer still remains unclear.

Adsorption of lysozyme to phospholipid and meibomian lipid monolayer films

It is believed that a lipid layer forms the outer layer of the pre-ocular tear film and this layer helps maintain tear film stability by lowering its surface tension. Proteins of the aqueous layer of the tear film (beneath the lipid layer) may also contribute to reducing surface tension by adsorbing to, or penetrating the lipid layer. The purpose of this study was to compare the penetration of lysozyme, a tear protein, into films of meibomian lipids and phospholipids held at different surface pressures to determine if lysozyme were part of the surface layer of the tear film. Films of meibomian lipids or phospholipids were spread onto the surface of a buffered aqueous subphase. Films were compressed to particular pressures and lysozyme was injected into the subphase. Changes in surface pressure were monitored to determine adsorption or penetration of lysozyme into the surface film. Lysozyme penetrated a meibomian lipid film at all pressures tested (max = 20 mN/m). It also penetrated phosphatidylglycerol, phosphatidylserine or phosphatidylethanolamine lipid films up to a pressure of 20 mN/m. It was not able to penetrate a phosphatidylcholine film at pressures ≥10 mN/m irrespective of the temperature being at 20 or 37 °C. However, it was able to penetrate it at very low pressures (<10 mN/m). Epifluorescence microscopy showed that the protein either adsorbs to or penetrates the lipid layer and the pattern of mixing depended upon the lipid at the surface. These results indicate that lysozyme is present at the surface of the tear film where it contributes to decreasing the surface tension by adsorbing and penetrating the meibomian lipids. Thus it helps to stabilize the tear film.

Surface Pressure Measurements of Human Tears and Individual Tear Film Components Indicate That Proteins Are Major Contributors to the Surface Pressure

Tear film stability has been associated with a low surface tension (high surface pressure), which has been attributed to a variety of tear film components. In this study, we examined the contribution of various tear proteins, mucin, and meibomian lipids to the surface pressure of human tears. A Langmuir trough was used to measure and compare the surface activities of albumin, lipocalin, beta-lactoglobulin, lactoferrin, lysozyme, secretory IgA, mucin, meibomian lipid, and tears. All proteins exhibited surface activity. The surface pressure-area (Pi-A) profiles of most protein films at equilibrium surface pressure (Pieq) were sigmoidal and showed hysteresis between the expansion and compression phases of the cycle. Pieq of most proteins took 4-9 hours to occur. By contrast, the Pi-A profiles for meibomian lipid films were hyperbolic rather than sigmoidal and had little hysteresis, and Pieq was attained within 1 hour. The Pi-A profiles of mucin films showed mostly hyperbolic characteristics with small hysteresis. The Pi-A profiles of films of tears were sigmoidal, showed strong hysteresis, and reached Pieq at about 5 hours. Partitioning of the proteins and whole tears into the subphase also occurred. Comparison between the dynamic Pi-A profiles of tears and those of individual tear film components shows that tear film proteins not only are capable of surface activity but also are major contributors to the surface activity of the tear film.