Surface Properties of Squalene/Meibum Films and NMR Confirmation of Squalene in Tears

Abstract

Squalene (SQ) possesses a wide range of pharmacological activities (antioxidant, drug carrier, detoxifier, hydrating, emollient) that can be of benefit to the ocular surface. It can come in contact with human meibum (hMGS; the most abundant component of the tear film lipid layer) as an endogenous tear lipid or from exogenous sources as eyelid sebum or pharmaceuticals. The aims of this study were to determine (i) if SQ is in tear lipids and (ii) its influence on the surface properties of hMGS films. Heteronuclear single quantum correlation NMR confirmed 7 mol % SQ in Schirmer's strips extracts. The properties of SQ/hMGS pseudo-binary films at the air/water interface were studied with Langmuir surface balance, stress-relaxation dilatational rheology and Brewster angle microscopy. SQ does not possess surfactant properties. When mixed with hMGS squalene (i) localized over the layers' thinner regions and (ii) did not affect the film pressure at high compression. Therefore, tear SQ is unlikely to instigate dry eye, and SQ can be used as a safe and "inert" ingredient in formulations to protect against dry eye. The layering of SQ over the thinner film regions in addition to its pharmacological properties could contribute to the protection of the ocular surface.

Keywords: Langmuir trough; NMR; meibum; sebum; squalene; tear film.

Figure 1

Structure of SQ. Numbering of carbons used throughout the text of this report.

Figure 2

¹H NMR spectra of tear lipids extracted from (i) Schirmer’s strip tear extracts (SSTE) or (ii) SQ. (A) =CH region; and (B) CH₃, CH₂ region. Numbers are the carbon numbers for SQ (Figure 1) assigned to the resonances.

Figure 3

¹H NMR spectra of tear lipid extracted from Shirmer’s strips atop the heteronuclear single quantum correlation (HSQC) spectra. Numbers correspond to the carbon numbers for SQ (Figure 1) assigned to the resonances. The chemical shifts are listed in Table 2. (A) CH resonance region; (B) Ester resonance region; and (C) CH₃, CH₂ resononance region.

Figure 4

Compression π(A)-isotherms of hMGS, squalene (curve 1 and 2–50 and 300 µg SQ deposited at the trough surface respectively) and SQ/hMGS mixtures. The weight percentage of squalene is shown on the figure legends. Upper panel displays compression isotherms when the amount of total lipid (=hMGS + SQ) on the surface is kept constant and the hMGS/SQ ratio is varied within this fixed total lipid quantity; and Bottom panel shows compression isotherms when the hMGS amount is kept constant, and the addition of SQ increases the total lipid amount on the surface.

Figure 5

Characteristic UltraBAM images (720 µm × 400 µm) of squalene layers, which shows irregular and partial spreading of squalene with lens formation already at high film areas; at compression, the lenses aggregate and get thicker.

Figure 6

Dependence of the maximal surface pressure of mixed hMGS/SQ films on the concentration of squalene (in weight %). Left panel shows the dependence when the amount of total lipid (=hMGS + SQ) on the surface is kept constant and the hMGS/SQ ratio is varied within this fixed total lipid quantity; and Right panel shows the dependence when the hMGS amount is kept constant, and the addition of SQ increases the total lipid amount on the surface. The π_max value depends on the “end members” of the surface film. If, at the end of compression, the interface between the lipid multilayer and the aqueous subphase is enriched with molecules with surfactant properties (e.g., polar lipids) the π_max value is high. If, at the end of compression, the interface between the lipid multilayer and the aqueous subphase is poor on molecules with surfactant properties (e.g., polar lipids) the π_max value decreases.

Figure 7

UltraBAM images (720 µm × 400 µm) of hMGS/SQ surface layers at various degrees of compression.

Figure 8

Upper panel: Typical stress-relaxation curve for film by equiweight meibum mixture (the subphase in the example is pure saline solution). The small compression deformation is completed in moment T and from this moment on the surface pressure starts to relax from a maximum value π_max to a new equilibrium value π_∞. The surface pressure relaxation transient is analyzed by fitting with the equation of double exponential decay shown to chart (see Equation (1)); and Bottom panels: Surface pressure relaxation transients of hMGS/SQ films. The data are presented in the format required for further fitting by double exponential decay Equation (1) as dependence of (π_t − π_∞)/(π_max − π_∞) on t. The time axis is normalized to begin from the start of the relaxation (the moment T). Data from stress-relaxation experiments with all hMGS/SQ films are summarized in Table 4.