where τr represents the characteristic time of viscoelastic relaxation, E∞ is the elasticity at time approaching infinity or storage modulus, and A1 is the amplitude of response to the initial surface perturbation. The response of each interfacial film to the area expansion and contraction was measured 10 times.
At this initial exploratory stage of the project, all the experiments were performed at ambient temperature, 22 ± 1°C.
Surface Tension Measurements
Surface tension of the reconstituted ex vivo lipid films was first studied as a function of film thickness. Fig. 2 shows that the surface tension of the lipid layers was reduced from 32 to 22.5 mN/m as the layer thickness increased from 2 to 15 nm and did not change with further thickening of the lipid film.
Fig. 3 shows the surface tension history at the air-saline interface after injection of lysozyme with continuous stirring of the aqueous phase; the working concentration of lysozyme in the cell in this experiment was 2 mg/ml. The surface tension decreased from 72.5 to 53 mN/m during the first hour, then continued to descend slowly for over 16 h. After 16 to 24 h of interface aging, the final tension was 50 ± 1 mN/m. This experiment was repeated for lysozyme concentrations ranging from 2.0 to 2.5 mg/ml, and the results (not shown here) suggested that the final surface tension was independent of the concentration. This observation was in agreement with data reported in the literature.4,5
In these experiments, after 16 h of equilibration, the aqueous lysozyme solution in the cell was displaced with buffered saline. The washout provided complete protein removal from the bulk phase inside the cell.30,31 The surface tension did not change after washout and remained 50 ± 1 mN/m, thus indicating an irreversible character of the protein binding to the air-water interface.
Fig. 4 illustrates the surface tension history of the mixed lipid-protein layer. An arrow indicates the moment when lysozyme was injected into the cell. Surface tension decreased by 1.5 mN/m immediately after lysozyme injection, then increased back to 26 mN/m and remained constant for another 10 h of measurements.
The results of the surface tension measurements during the injection, 30-min equilibration, and then washout of opti-free express (OFX) are presented as a function of time in Fig. 5. In this set of experiments, an air bubble was formed in 19 ml of buffered saline with a surface tension of 72.5 mN/m. A few minutes later, as indicated by the arrow in Fig. 5, 1 ml of OFX was injected into the cell with continuous stirring for 30 min. The surface tension dropped to approximately 40 mN/m almost instantaneously upon OFX injection. Continuous stirring and buffer flow through the cell caused the scatter (±1 mN/m during washout) of the surface-tension data during the washout period shown in Fig. 5. Before washout, stirring was stopped for 2 to 3 min, and the surface tension at the air-OFX (1:20 dilute) interface was found to be 36.5 ± 0.5 mN/m. This value was in agreement with that reported by Dalton et al.38 for undiluted OFX. Stirring was then resumed, and 500 ml of buffered saline was pumped through the cell. The final tension at the air-saline interface increased by 3 mN/m after OFX was washed out from the bulk phase but still remained low at 39.5 ± 0.5 mN/m. Similar behavior was observed upon injection and washout of opti-free replenish (OFR). The surface tension for OFR (1:20 dilute) was 35.2 ± 0.5 mN/m, which then increased to 38.5 ± 0.5 mN/m after washout. The surface tension at the air-aqueous interface after a complete washout of both OFX and OFR from a bulk solution remained much lower than 72.5 mN/m, which is typical for a pristine air-water interface. These observations suggested that both MPSs used in this study contained some irreversibly adsorbing components, which remained attached to the interface after bulk-surfactant washout.
Fig. 6 illustrates the surface tension history of the mixed lipid-protein layer that was exposed to OFX for 30 min then rinsed with buffered saline. Injection of OFX into the cell caused a slight decrease (∼1.5 mN/m) of the mixed film surface tension. After displacement of the aqueous phase with pure buffered saline, the surface tension returned to its original value of 26.1 ± 0.3 mN/m. The same trend was observed upon injection of OFR followed by washout with buffered saline. Unchanged surface tensions indicated that MPS did not displace, solubilize, or destroy the predeposited ex vivo lipids during the 30-min exposure. However, these observations do not provide sufficient information regarding specific interactions between MPS and the mixed lipid-protein films.
To acquire more information in relation to the interactions between MPS and lipid-protein complexes, we used the interfacial step-strain relaxation method to study in detail the rheological properties of the layers formed by each of these substances alone and of the mixed interfacial layers. Rheological parameters are often used to characterize the interfacial properties of thin films under dynamic conditions and are relevant for many practical applications where the interfacial film stability is involved.33,35–37
Interfacial Elasticity Measurements
The interfacial viscoelastic behavior of the individual layers formed by lysozyme and the reconstituted ex vivo lipids was studied. Fig. 7 shows the interfacial elasticity history of the layer formed by lysozyme (bulk concentration 2.5 mg/ml), before (Fig. 7a) and after (Fig. 7b) washout of protein from the bulk solutions, which served as control experiments for further studies of MPSs influence on the interfacial elasticity of mixed lipid-protein films.
Fig. 7a shows that before washout (i.e., in the presence of lysozyme in a bulk solution), the responses of the interfacial protein layer to expansion and contraction of surface area completely coincide. The single-exponential decay model (Eq. 2) was fit to the experimental data, and rheological parameters were thus estimated. E∞ was found to be 25.6 ± 0.5 mN/m, and τ was 460 ± 20 s for both expansion and contraction of the protein interfacial layers.
As seen in Fig 7b, protein washout from the bulk phase changed the viscoelastic response of the interfacial layer, and for postwash dilatations, some hysteresis arose between the expansion and contraction steps. E∞ increased after washout, becoming 34.5 ± 0.5 mN/m and 33.2 ± 0.5 mN/m for expansion and contraction, respectively. The postwash relaxation time τ also increased from 460 to 760 ± 10 s and 630 ± 15 s for expansion and contraction, respectively.
Fig. 8 depicts typical transient elasticity vs. time curves for the interfacial layers formed by reconstituted ex vivo lipids (curve 1), for the same lipids after injection and equilibration of this layer with lysozyme (curve 2), and after washout of the mixed layer (curve 3). Elastic response of the lipid layer alone was independent of whether expansion or contraction was applied, i.e., no hysteresis was observed. E∞ of the ex vivo lipid layers was close to that of the lysozyme, 25.3 ± 2.5 mN/m; τ for the lipid multilayers was 90 ± 25 s, 6 to 7 times shorter than that of the lysozyme layers. For the mixed ex vivo lipid-lysozyme interfacial films, E∞ was found to be 24.8 ± 3.7 mN/m; it did not change upon lysozyme adsorption at the lipid-aqueous interface. This result is as expected, as both of the components, lipids and lysozyme, have similar values of E∞. However, the relaxation time τ for the mixed lipid-protein films was 250 ± 35 s, 2.7 times higher than for the lipids alone and 1.8 times lower than for lysozyme alone. In the control run (curve 3), where a lipid-protein mixed film was washed with buffered saline without exposure to MPS, E∞ increased slightly by 2 ± 1.2 mN/m, after protein removal from the bulk phase.
Fig. 9 depicts an example of the transient elasticity of a postwash OFX interfacial layer at the air-water interface, without preadsorbed lipids or protein. One can see from this plot that the interfacial rheological behavior of postwash OFX was distinctly different from that of the lipids, protein, or the mixed lipid-protein layer. The OFX layer exhibited a threefold lower E∞ than lysozyme or lipids. For both postwash OFX and OFR layers, E∞ was 8.3 ± 0.5 mN/m. In contrast, τ was 950 ±50 s for the postwash MPS layers; these layers relaxed 2 times slower than protein and 9.5 times slower than the lipids. MPS formed relatively weak, viscous layers relaxing slowly after expansion or contraction of the interface.
Fig. 10 shows the elasticity history of the mixed lipid-protein layers first exposed to either OFX or OFR for 30 min, then washed with buffered saline. MPS induced noticeable changes in the viscoelastic behavior of the mixed lipid-protein layers. After exposure to OFX, the postwash E∞ of the mixed layers was reduced from approximately 25 ± 2 to 13 ± 4.5 mN/m (p < 0.001, pair t test), almost equal to the value observed for postwash OFX alone. The τ of the films treated with OFX increased from 250 to 450 ± 30 s (p < 0.001, pair t test), suggesting that some of OFX remained adsorbed onto the initial predeposited lipid-protein layer.
Exposure of the mixed lipid-protein films to OFR produced a weaker effect on their viscoelastic behavior. The E∞ of the mixed layers was reduced from 25.6 to 21 ± 3.0 mN/m after 30-min exposure to OFR (p = 0.004, pair t test). In contrast, τ increased almost threefold to 730 ± 55 s (p < 0.001, pair t test). Once again, these results suggest that the surface-active components of MPS partially displaced the lysozyme and became irreversibly attached to the predeposited lipid-protein layer.
Previous investigations of human tear constituents and whole tear surface properties such as surface tension have shown that the tear lipid layer plays a significant role in tear film surface tension reduction and tear film stability.4–6,10,11 McCulley and Shine15,16 have developed a composition-based model for the thick tear lipid layer, proposing a complex stratified multilayered structure. According to this model, polar amphiphilic lipids (mostly phospholipids comprising 6 to 16 percent of the total lipids) with surfactant characteristics serve as a structure upon which the stability of tear lipid film is largely dependent, although most of the tear lipid film consists of non-polar components (84 to 94%). The thickness of the tear lipid layer is one of its most extensively studied properties, and the estimates range from 13 to 100 nm depending on the method used for measurements.14,17 These measurements were consistent with the thick multilayer model of McCulley and Shine.15,16 The thickness of a dipalmitoyl phosphatidyl choline (DPPC, a phospholipid, which is the major constituent of pulmonary surfactant and is most commonly used as a model for tear lipids) monolayer was in the range 2 to 2.5 nm as determined by neutron reflectivity measurements.22 Even at the lowest estimated thickness of 13 nm, the tear lipid film is at least 6 times thicker than a monolayer; therefore, monolayers of meibomian lipid previously investigated may not be an adequate model of the human tear lipid layer. In a subsequent investigation, Shine and McCulley39 conducted a detailed study of polar lipids in human meibomian secretion and suggested that the polar lipid layer was most likely 1 to 3 molecules thick and served as a surfactant between the aqueous tear and the nonpolar part of the lipid layer. Recently, Butovich et al.17,34 summarized the most current data regarding the structure and chemical composition of meibomian and tear lipids. The authors proposed that tear lipids form a thick blanket, with a minimum thickness of 20 molecules, over the aqueous layer of the tear film.
The interfacial pressure of meibomian lipid monolayers has been examined in a number of articles.6,18–22 In these publications, lipid monolayers were chosen to model the tear lipid layer and to investigate in greater detail the interaction of lipid monolayers with model tear proteins. Tragoulias et al.20 found that model tear proteins possessed significant surface activity; from these results, the authors concluded that tear proteins were the major contributors to the surface pressure of a tear film. Several studies18–20 have explored monolayers of meibomian lipids at relatively low surface pressures corresponding to low surface coverage by polar lipids. Under these conditions, proteins were able to penetrate into the lipid monolayer and reduce the surface tension.18
In this study, we focused on the interfacial properties of relatively thick ex vivo human lipid multilayers. We believe that the behaviors of thick lipid films provide a more realistic model of the human tear lipid layer than the lipid monolayers studied previously. The results shown in Fig. 2 clearly indicate that surface tension strongly depended on ex vivo lipid layer thickness. The thinnest lipid film we studied was a 2 nm thick monolayer. The surface tension of this layer was close to 32 mN/m, corresponding to the surface pressure of 40 mN/m reported by other researchers.18–20 However, as the thickness of the deposited lipid layers increased, the surface tension decreased until it reached a constant value of 22 ± 1 mN/m at a lipid layer thickness of 15 nm or greater. These results suggested that the saturation, or coverage, of the lipid-aqueous interface with polar lipids increased in concert with the total ex vivo lipid film thickness. At a total lipid thickness between 15 and 25 nm, when a completely saturated, densely packed polar lipid layer was formed, the films exhibited a very high surface pressure of ∼ 50 mN/m, which is in agreement with the values found for condensed phospholipid layers and lung surfactants.40,41 The important implication for lipid film dynamic behavior in vivo arises from these observations. The curve in Fig. 2 can be dissected into two linear parts: one for thin films between 2 and 10 nm, and the second for films thicker than 20 nm. The first part of the linear fit gives a surface tension gradient, dγ/dh = 1.0 (mN/m)/nm, whereas for the second part, the tension gradient is 30 times lower, 0.033 (mN/m)/nm. The tension gradients are known to be the driving force for the mass transfer along a liquid-air interface, which causes flows in liquid films. This phenomenon is known as the Marangoni effect. In thick lipid films, small tension gradients lead to sluggish flows and slow film thickness rearrangement. Therefore, clinically observed interference patterns24 persist in the case of a thick lipid film. However, in thinner lipid films, a slightest decrease in the film thickness gives rise to high surface tension gradients, resulting in strong Marangoni flows, which lead to thickness reequilibration.
As shown in Fig. 4, lysozyme injected into the aqueous phase surrounding an ex vivo lipid film did not significantly change the surface tension at the air-aqueous interface even after long-time equilibration with continuous stirring. This observation was consistent with the results reported by Miano et al.22 for a mixed DPPC-lactoferrin layer at an initial DPPC surface pressure of 35 mN/m or higher. In that study, neutron reflection measurements were performed on a DPPC-lactoferrin layer, and it was found that at high surface pressures, protein adsorption into the lipid layer was very low. The results of our surface tension measurements led us to the conclusion that lysozyme, similar to lactoferrin, could not penetrate into or displace the condensed, thick layer of reconstituted ex vivo lipids. The evidence of lipid-lysozyme interaction and formation of an interfacial lipid-lysozyme complex in this study was obtained using an interfacial relaxation technique in combination with a washout option.
We found significant differences between the interfacial rheological parameters of the ex vivo lipid layers alone and of the mixed lipid-lysozyme layers. These differences persisted even after complete protein removal from the bulk phase, indicating that lysozyme was irreversibly bound to the lipid layer. At high surface pressures, the presence of lysozyme in the mixed interfacial layers was undetectable by surface tension measurements. However, it manifested itself by an approximately twofold increase in mixed film relaxation time compared with that of a layer formed by ex vivo lipids alone. The interfacial rheological properties of the reconstituted ex vivo lipids in the thick mixed layers were much more sensitive indicators of their interaction with protein than were surface tension or surface pressure. Changes in interfacial rheological parameters after protein washout were most likely caused by the removal of loosely bound protein molecules from the adsorbed interfacial layer, leading to increased interfacial viscosity.
The adsorption of lysozyme into the ex vivo lipid layers was likely driven by electrostatic interactions. Lysozyme has an isoelectric point of 11 and a net positive charge at pH 7.3. Polar tear lipids are known to contain a significant amount of the species with negatively charged head groups, such as anionic phospholipids and fatty acids.15,17 These anionic head groups are likely to interact with the positively charged groups of the lysozyme molecules, forming complexes through an ion-pairing mechanism.
Lysozyme is one of the most abundant proteins in human tears.25,26 We hypothesize that the binding of lysozyme molecules to anionic lipid head groups may play a very important role in maintaining tear film integrity. Lysozyme and other tear proteins, coupled with polar lipids in the tear lipid film, may provide additional tethering and stronger linkage between the hydrophobic lipid layer and the underlying tear aqueous.
MPS, with its relatively high concentration (0.1% by weight) of Tetronic T1304 and other surface-active ingredients reduced the surface tension at the air-aqueous interface by ∼32 to 34 mN/m. Our previous studies of Pluronics (nonionic surface-active polyethylene-polypropylene block copolymers) adsorption at air-water and water-mineral oil interfaces have shown that these polymers bind irreversibly to the interfaces.32 At the water-oil interface, the polymeric surfactants were found to compete successfully with a synthetic double-tailed surfactant Aerosol OT (bis-ethyl-hexyl sulfosuccinate, sodium salt), whose structure resembles that of natural phospholipids.33 Tetronic T1304 also belongs to the family of nonionic surface-active polyethylene-polypropylene block copolymers. Several experiments conducted with aqueous solution containing only 0.1 wt% of Tetronic T1304 have revealed that, similar to Pluronics, Tetronic binds irreversibly to the air-aqueous interface. We hypothesize that in MPS, Tetronic T1304 is responsible for an irreversible reduction of surface tension at the air-solution interface. Moreover, interfacial rheology studies conducted with mixed lipid-lysozyme layers exposed to MPS (Fig. 10) have shown that both OFX and OFR alter the rheological characteristics of mixed lipid-lysozyme films. The changes caused by OFX were found to be significant and persistent, whereas OFR produced milder effects. Besides Tetronic T1340 and other components present in both OFX and OFR, OFX contains an unspecified amount of AMP 95, amino propanol, which is a polar organic solvent, whereas OFR contains nonanoyl ethylenediaminetriacetic acid, which is a surface-active fatty acid. The differences in formulations between OFX and OFR, slight as they may be, were the major reason for the differences in MPS performance. It is conceivable that MPS cytotoxicity42 might be associated with high surface activity of some ingredients and their interaction with the phospholipids on corneal epithelium cells.
The results of this study show that MPS surface tension (Fig. 5) was at least 10 mN/m lower than that of lysozyme (Fig. 4), suggesting that MPS contains ingredients that are more surface active than lysozyme. The interfacial layers became more plastic after treatment with OFX (Fig. 10), indicating that OFX partially displaced protein from the mixed lipid-protein interfacial film. The difference in the responses of lipid films to exposure to OFX and OFR suggests a new explanation for documented differences in their cytotoxicities.42 It seems likely that OFX ingredients interacting with phospholipids comprising cell walls might compromise the cell-wall protective function by making the lipid membranes more permeable to potentially cytotoxic preservatives. The milder disruptive effect of OFR on lipid-protein mixed films correlates with its lower cytotoxicity level.
Tear films are complex, multilayered biological colloid systems. The composition of the tears is delicately balanced and regulated by ocular tissues to maintain optimal function under the high stress exerted by the eyelids during blinking. It is reasonable to assume that the interfacial rheological properties of healthy tear films are also optimally adjusted to provide timely and full recovery after very rapid compression (closed eye) followed by expansion (open eye). Soaps and shampoos contain highly concentrated surfactants that are much more surface active than tear proteins. These substances are able to solubilize and destroy tear lipid films,1 causing the familiar, extremely unpleasant sensation when soap or shampoo comes into contact with the ocular surface. As we have shown above, MPS containing some highly surface-active ingredients was able to displace lysozyme from mixed ex vivo lipid-protein layers, leading to alterations of lipid-protein film interfacial rheological characteristics. In an eye, the delicate natural balance of tear film interfacial properties might become compromised by MPS leaching from a lens. There are indications in the literature that highly porous soft pHEMA-MAA lenses (Acuvue 2), immersed in solution overnight, were able to accumulate up to 36 μg/lens of Tetronic and then release this surfactant into a buffered saline.43,44 A similar process may occur when a contact lens wearer inserts such a lens on an eye after overnight storage in MPS. The surface active components trapped inside the lens matrix may be slowly released into the eye, interacting with the mixed tear lipid-protein films and displacing proteins from this layer. Such displacement might produce undesirable alterations of tear film interfacial rheological properties, reduced tear film stability, and discomfort after several hours of lens wear. These findings help to explain the reduced tear film thickness and discomfort reported by patients who used OFX.12 It should be emphasized that in clinical practice, special measures must be taken to find a proper combination of lens material and lens care solution that will be the safest, most suitable, and comfortable for each patient, especially for those with compromised tear film stability and short tear break-up time.
We thank C. J. Radke, PhD, Department of Chemical Engineering, University of California, Berkeley, for providing laboratory space and access to equipment. We also thank H. Green, OD, PhD, T. Sanders, OD, and A. Graham, MS, for their valuable feedback during manuscript preparation.
Meng C. Lin
Clinical Research Center
School of Optometry
University of California
Berkeley, California 94720-2020
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Keywords:© 2010 American Academy of Optometry
ex vivo human tear lipids; tear proteins; soft lenses; lens care solution; lens induced dryness