The Adsorption of Major Tear Film Lipids In Vitro to Various Silicone Hydrogels over Time

Abstract

purpose. An in vitro study was conducted to measure the adsorption of major tear film lipids to soft contact lenses over time.

methods. Commercial balafilcon A (PureVision; Bausch & Lomb, Rochester, NY), galyfilcon A (Acuvue Advance; Johnson & Johnson Vision Care), lotrafilcon A and B (Night & Day And O₂Optix; CIBA Vision, Duluth, GA), senofilcon A (Acuvue Oasys; Johnson & Johnson Vision Care, Jacksonville, FL) and etafilcon A (Acuvue 2; Johnson & Johnson Vision Care) lenses were all soaked for 14 hours in the dark at 34.5°C in either cholesterol (CH; nonpolar lipid) or phosphatidylethanolamine (PE; polar lipid), tagged with 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) and N-fluorescein-5-thiocarbamoyl (FITC) labels, respectively. After rinsing, the lenses were measured for fluorescence and the corresponding lipid concentration was calculated from an appropriate standard curve. The lenses were then placed into a fresh 1-mL aliquot of the lipid being tested, and the procedure was repeated for 20 days.

results. In vitro adsorption of CH was greater that that of PE for all lens types (P < 0.0001 at days 14 and 20). After 20 days of soaking in PE, the lotrafilcon polymers showed the lowest adsorption of all the silicone hydrogel lenses tested at 0.4 and 1.5 μg/lens, for lotrafilcon A (P ≤ 0.0001) and lotrafilcon B, respectively (P ≤ 0.0001). Galyfilcon A and senofilcon A showed significantly higher PE adsorption at 5.1 and 4.9 μg/lens, respectively, compared with all other silicone hydrogel lenses investigated (P < 0.03). Senofilcon A (P < 0.0001) and balafilcon A (P < 0.02) had the highest affinity for CH of all the lens types after 20 days, with adsorptions of 23.2 and 24.1 μg/lens, respectively. Lotrafilcon B (P < 0.0001) showed the lowest in vitro adsorption of CH of all the lens types, at 3 μg/lens.

conclusions. In vitro lipid adsorption varied greatly depending on the lens material for both polar and nonpolar lipids. Overall, there was less in vitro adsorption of lipid to the lotrafilcon A and B polymers than for any of the other silicone hydrogel polymers tested. The quantity of lipid adsorption by lotrafilcon polymers was similar to “conventional” hydrogel lenses.

Spoilage of contact lenses by either proteins or lipids is an important factor in the biocompatibility of contact lens materials. ¹ Such fouling may produce unfavorable effects on the function of the contact lens as well as the wearer’s experience. Some of these effects may include tear film disruption, decreased vision, discomfort, intolerance, and bacterial adhesion. ^{2 3 4 5 6 7} Silicone hydrogel lenses are composed of a new family of materials unlike any other contact lens material used. ⁸ They have been shown to adsorb lower amounts of protein than conventional hydrogel materials; however, lipid adsorption has been reported to be more significant. ⁸  

The tear film lipid layer, which is thought be between 1% and 9.5% of the tear film, ^{9 10 11} is formed primarily from lipids secreted by meibomian glands. ^{9 12 13 14} The lipid layer protects the aqueous tear fluid from evaporation and provides a more stable and smooth tear film for refractive properties, retard evaporation of the aqueous tear, lubricates during the blink, and act as a barrier to foreign bodies. ^{12 13 15 16} This layer is composed of two phases: a thin polar phase that is adjacent to the aqueous-mucin phase of the tear fluid and a thick nonpolar phase that is associated with both the polar phase and the environment. ^{17 18 19 20} The polar phase, which is 8% of the lipid layer, consists of 70% phospholipids, ⁹ which are predominantly phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelins, and unknown. ^{9 21 22} Greiner et al. ²¹ found that most polar lipids are constituted of PE and PC which together comprise nearly 60% of phospholipids. The nonpolar phase, which is 92% of the lipid layer, ⁹ consists of mainly cholesterol (CH) and CH esters. ^{9 12 17 18} Although the nonpolar phase protects the aqueous tear fluid from evaporation, ^{12 16} providing a more stable and smooth tear film for refractive properties, ¹⁵ the polar phase is the structure which the integrity of the nonpolar phase depends, acting as an interface between the aqueous tears and nonpolar lipid ^{17 21} and may have greater propensity to be in direct contact with the contact lens material. 

Maissa et al. ²³ showed that hydrogel lens wear changed the tear film lipid composition by decreasing the polar lipids and increasing nonpolar lipids. In decreasing polar lipids the tear film would become less stable therefore increasing the rate and duration of wetting and drying cycles and perhaps increasing deposition by lipids and other tear components. Historically, most adsorption data reported have been for nonpolar lipids, specifically CH, probably because of their greater presence in the tear film compared with polar lipids and on worn contact lenses. ^{7 9 19 24} Lipid adsorption may be a more prevalent issue for SiHy lenses compared with traditional HEMA lenses because of the relatively hydrophobic surfaces. ²⁵ This may cause polar lipids to play a more important role than previously thought. This study investigates a comparison of adsorption in vitro between polar and nonpolar lipids. There is a great deal of literature surrounding the concentrations of lipid and lipid species within the tear film. ^{9 12 18 20 21} Through consulting the literature we chose a polar and nonpolar lipid to investigate in this study. These were decided on because of their reported presence in the tear film and the commercial availability of these lipids with fluorescent tags attached. The fluorescent tag allows for continual detection without extraction or destruction of lenses. The representative nonpolar lipid chosen was CH, and the polar lipid chosen was PE. We attempted to mimic physiological concentrations by averaging the percentage presence of both the tear film layer and the percentage presence of the individual lipids reported in the literature. ^{9 10 11 17 18 20 21 26 27}  

Materials and Methods

Lenses used in this study were all commercially available silicone hydrogel polymers except etafilcon A (Acuvue 2; Johnson & Johnson Vision Care, Jacksonville, FL) which is a commercially available conventional HEMA (hydroxyethylmethacrylate) contact lens. All lenses assayed were −1.00 D. A low power was chosen to minimize any background fluorescence from the sample. The silicone hydrogel lenses used were balafilcon A (PureVision; Bausch & Lomb, Rochester, NY), galyfilcon A (Acuvue Advance; Johnson & Johnson Vision Care), senofilcon A (Acuvue Oasys; Johnson & Johnson Vision Care), lotrafilcon A (Night & Day; CIBA Vision, Duluth, GA), and lotrafilcon B (O₂Optix; CIBA Vision). Table 1details some of the characteristics of these polymers. 

PE labeled with FITC ([N-fluorescein-5-thiocarbamoyl], PE-FITC; Invitrogen-Molecular Probes, Carlsbad, CA) and CH labeled with NBD ([7-nitrobenz-2-oxa-1,3-diazol-4-yl], CH-NBD; Avanti, Alabaster, AL) were the lipids chosen for investigation in this study. 

Experimental Design

A separate standard curve was set up in duplicate for each lens type under investigation for both PE-FITC and CH-NBD. The lipids were solubilized in a stock solution of 1 mg/mL lipid in methanol at 34°C. Aliquots were taken from this stock to make standard curves in phosphate-buffered saline (PBS) at pH 7.4 in a concentration range from 0 to 20 μg/mL for both lipids. All solutions were made up at a temperature of 34°C and checked under 10 times magnification (5MZ-2T; Nikon, Tokyo, Japan) to determine solubility. One milliliter of standard at each concentration was placed in the well of a 24-well plate (Costar 3473; Corning Corp., Corning, NY). An appropriate lens was also placed in each well of the standard curve to correct for any autofluorescence produced by the lens polymer itself. 

Five lenses of each type were placed in another 24-well plate and soaked alongside the standard curve samples in 1 mL of a concentration that mimicked physiological concentrations as closely as possible. As mentioned earlier, these concentrations were determined by averaging reported percentages of their presence from the literature. ^{9 10 11 17 18 20 21 26 27}  

These were determined to be 0.5 μg/mL PE (PE-FITC) and 1.75 μg/mL of CH (CH-NBD). All concentrations were made up in PBS at pH 7.4. Standard curves and test plates were all wrapped in aluminum foil to maintain darkness and were incubated for 14 hours, with rocking, to mimic an average day’s wear of contact lenses at an ocular temperature of 34.5°C. ²⁸  

After 14 hours the standard curve and test plates were removed from the incubator. The standard curve plates were immediately read on a multilabel fluorescence counter (Wallac Victor II 1420; PerkinElmer Life and Analytical Services, Waltham, MA) at a wavelength of 465 nm. Although this is not the manufacturer-specified optimum wavelength for reading both FITC and NBD, it was the optimum wavelength for balancing good detectability for the fluorophore with minimal noise from the background fluorescence of some of the lens polymers. The test plates were washed three times in 1 mL of PBS, to ensure that only bound lipid would be determined without soaking fluid carryover due to surface tension. This method allowed for material affinities to be assessed more accurately. The lenses were placed in a fresh 24-well plate containing 1 mL of PBS in each well and read on the fluorescence counter at 465 nm. After the test samples were read, the PBS was removed, and 1 mL of a fresh solution of either PE-FITC or CH-NBD were placed on the lenses in the same concentrations as previously mentioned and placed back in the incubator at 34.5°C, with rocking, until the next period. Although it would have been interesting to be able to mimic the longest wear time possible for some of these polymers which is approximately 30 days, this procedure was repeated for 20 days consecutively and could not be continued past this time, as a decay in the fluorescence signal started to occur, creating compromised linearity in the standard curves. The fluorescence counts for each lens were calculated in micrograms per lens from the standard curve measured that day. The entire protocol was repeated with another five lenses of each type, to produce a total of 10 lenses for each lipid type for each day. 

Statistical analysis was performed with Student’s t-test. The two-sample t-test was run to assess all pair-wise differences among the six brands. Because of the small sample size compared with the number of time points this was the most robust method of analysis. 

Results

Overall, the in vitro adsorption of CH was greater than that of PE for all lens types, including etafilcon A (day 14 P < 0.0001, day 20 P < 0.0001; Figs. 1 2 ). Averaged over the 20 days of the study, the silicone hydrogel lenses CH adsorption was higher than that of PE by 3.7- to 25.1-fold and was dependent on lens type. In vitro adsorption of both polar and nonpolar lipids appeared to reach saturation with galyfilcon A in approximately 12 to 14 days. In vitro adsorption to lotrafilcon A and lotrafilcon B lenses plateaued at approximately day 17. For both senofilcon A and balafilcon A, in vitro saturation of adsorption of PE occurred by day 14, whereas saturation was not complete with CH by day 20. 

CH Adsorption

Balafilcon A, etafilcon A, and senofilcon A lenses had significantly higher in vitro adsorption of CH than did lotrafilcon B lenses after day 1 at physiological concentrations (P < 0.002). By day 4, adsorption of CH to balafilcon A (P < 0.002) and the lotrafilcon A (P < 0.002) and lotrafilcon B (P < 0.0001) polymers were significantly lower than all other lenses tested, with lotrafilcon B adsorbing significantly less than any of the other lenses (P < 0.003). 

Day 14 showed a dramatic increase in adsorption to balafilcon A (13.9 ± 3.2 μg/lens), similar to the levels for senofilcon A and galyfilcon A (18.8 ± 2.3 and 15.03 ± 1.3 μg/lens, respectively) lenses. In contrast, lotrafilcon A showed adsorption similar to that of the traditional HEMA lens, etafilcon A (5.9 ± 1.3 and 6.5 ± 1.7 μg/lens, respectively). Lotrafilcon B again showed significantly lower adsorption than all other polymers (2.6 ± 1.1 μg/lens) with lower adsorption than a traditional lens such as etafilcon A (P < 0.0001). This rank order of adsorption carried through to day 20 when balafilcon A, senofilcon A, and galyfilcon A exhibited at least 1.5 times more CH adsorption than lotrafilcon A and etafilcon A and up to 6 times more than lotrafilcon B (Fig. 1) . 

PE Adsorption

After 1 day of exposure to this polar lipid, etafilcon A (P < 0.03), lotrafilcon A (P < 0.03), and lotrafilcon B (P < 0.03) lenses showed significantly lower adsorption than all other lens types tested. This trend followed through to day 20, with etafilcon A and lotrafilcon A and B all showing low binding (0.1 ± 0.1, 0.4 ± 0.2 and 1.5 ± 0.5 μg/lens respectively). Galyfilcon A (P < 0.03) and senofilcon A (P < 0.01) showed a significantly higher degree of in vitro PE adsorption at 5.1 and 4.9 μg/lens respectively. Balafilcon adsorbed 3.2 μg/lens of PE. 

Compared with lotrafilcon B, in vitro adsorption of PE was 6 times greater for galyfilcon A and 3.5 times greater for senofilcon A and balafilcon A lenses on day 14. By day 20 the ratio of the difference between these polymers’ adsorption to lotrafilcon B was at least a factor of two. In vitro adsorption of PE on days 14 and 20 to senofilcon A, galyfilcon A, and balafilcon A lenses was in excess of 3.5 times more than that to lotrafilcon A lenses. 

Although all polymers were shown to be statistically different from each other in adsorption of PE, the lotrafilcon polymers exhibited adsorption levels closer to the range found with the HEMA-based hydrogel tested (Fig. 2) . 

Discussion

Silicone hydrogel contact lenses exposed to different lipids under identical and controlled conditions have significantly varying affinities as a function of both lens and lipid types. Although all lenses showed a detectable level of lipid adsorbed, there was a greater affinity for the nonpolar lipid (CH) than for the polar lipid (PE) selected for this study. This result may indicate that the ionicity of a lipid does not play a significant role in its affinity to a material. However, the driving mechanism of the lens to attract lipid is not as clear. 

Research performed on lipid adsorption to hydrogels has examined the roles of ionicity and water content in the attraction of lipids. Botempo and Rapp ²⁹ conducted a study in which they found that high-water, nonionic lenses (group II) were more prone to lipid deposition, and low-water, ionic lenses (group III) were the least prone to fouling of lipids. They also concluded that nonionic lenses (groups I and II) adsorb more lipid than ionic lenses (group III and IV). This theory was reinforced by a study conducted by Jones et al. ³⁰ These studies focused primarily on traditional HEMA-based hydrogels and not the newer silicone hydrogel materials currently in the market. The present study focused on silicone hydrogels, and the adsorption data did not fit the expected profile discussed in historical studies, ^{29 30} which may suggest that silicone hydrogels do not fit neatly into the de facto FDA lens groups previously established for traditional hydrogels. 

The highest amount of adsorption overall was with senofilcon A, galyfilcon A, (group I lenses), and balafilcon A (group III lens), whereas the lowest adsorption was with lotrafilcon A and B (group I lenses) and etafilcon A (group IV lens). Based on research by Botempo and Rapp ²⁹ it would be expected that balafilcon A lenses would have lower adsorption of lipid than all the other lens types tested (groups I and IV). It should be noted that etafilcon A was the group IV and traditional lens chosen in this study that showed a low affinity for lipid adsorption. This profile may have been different if a group IV HEMA material with more hydrophobic monomers, such as vifilcon A, had been chosen. 

After day 1, PE adsorbed to balafilcon A, galyfilcon A, and senofilcon A lenses in greater amounts than to lotrafilcon A and B and etafilcon A lenses. This difference in adsorption between the polymers continued to day 20. The large disparity in lipid adsorption among the group I lenses may be due to water content, as adsorption increases with lens water content. Lotrafilcon A and B polymers have a water content of 24% and 33%, respectively, and senofilcon A and galyfilcon A with 38% and 47%, respectively. Another contributing factor could be the ability of pyrrolidone derivatives to solubilize lipids. ^{30 31} Senofilcon A and galyfilcon A both contain PVP in the reaction mixture, whereas the lotrafilcon polymers do not. 

CH showed a different adsorption pattern early in the time course, with etafilcon A having greater adsorption on day 1 than all other lens types and lotrafilcon B having significantly lower adsorption than all other lens types. However, this pattern changed by day 8, with balafilcon A, senofilcon A, and galyfilcon A all having significantly higher adsorption than other lens types investigated. 

Jones et al. ²⁵ reported that lipid adsorbed in higher amounts to balafilcon A compared to lotrafilcon A and etafilcon A, although the amount detected in that study was approximately 10 times higher than detected in this study with the same lens types, perhaps because of the difference between in vitro and ex vivo studies. The protein-lipid interactions and drying and wetting cycles of the lens during wear may significantly increase the amount adsorbed by all lenses. 

To date, commercially available silicone hydrogel lenses benefit from a surface treatment to enhance wettability by the tear film. ³² Theses lenses’ hydrophobic nature and the loose molecular structure of silicone makes them lenses permeable to lipid soluble materials. ³³ To make these materials more biocompatible, a surface treatment was used. ³²  

The lotrafilcon A and B polymers have been treated to provide a 25-nm plasma polymerization surface treatment with a high refractive index. Balafilcon A uses a plasma oxidation treatment rendering the surface with glassy, discontinuous silicate islands ³² and increasing the contact angle of the surface compared with lotrafilcon A. ⁸ Senofilcon A and galyfilcon A have a different type of surface treatment, as the polymer formulation contains Hydraclear (Johnson & Johnson), a PVP compound in the reaction mixture that provides a hydrophilic PVP surface treatment disguising silicone elements of the lens surface. ^{34 35}  

The differences in these surface treatments may be the key to explaining their different affinities to lipid as well as to when and why the lipid adsorption plateaus or decreases for these lenses, as imperfections in the lens surface is where deposits are likely to form. ³⁶ Establishing a consistent and biocompatible surface on silicone hydrogels may have an advantage other than producing hydrophilicity. The lens may become more resistant to lipids and lipoproteins. ³³ Any heterogeneity in this surface, as is seen with balafilcon A, ³² may promote increased lipid fouling in these regions of hydrophobicity. ³⁷ When the surface is not continuous or biocompatible enough to retard lipid adsorption the chemistry of the bulk may also play a role as NVP (N-vinyl pyrrolidone), which balafilcon A contains (Table 1) , has been shown to have a significant affinity to lipid. ³⁰ Also galyfilcon A and senofilcon A contain a pyrrolidone derivative as an internal wetting agent (PVP; polyvinyl pyrrolidone) that may cause increased lipid solubility, ³¹ thus providing them with a greater propensity for lipid sequestering. 

In this study, it was possible to observe the adsorption of lipid showing steady increases, decreases, and a plateau in adsorption over time. The course differed for each lens type and lipid investigated. Sharp decreases that rose again the next day, as was seen frequently for PE with senofilcon A and balafilcon A after day 12, may be explained by the rinsing step in the assay each day. This process may have acted to clean the lens. However, the steady decrease in CH observed with etafilcon A after day 16 may be due to the saturation of binding sites and the steady release of reversibly bound lipid. However, when lipid adsorption plateaued for lenses at various times, such as at day 12 with galyfilcon A for CH, it is likely an indication of the saturation of binding sites by irreversibly bound lipid. 

Overall, the data indicate that the deposit characteristics of silicone hydrogels do not always behave as expected by the current FDA lens material grouping. Although this study supports the current literature, in that water content and ionicity of the lens played a role, it also puts forth a new discovery that is more pertinent to silicone hydrogels. The chemistry of a surface treatment and homogeneity of the surface are vital as the first line of defense against fouling of silicone hydrogel lenses by lipids. 

 Submitted for publication March 29, 2007; revised August 24 and October 11, 2007; accepted November 28, 2007.

 Disclosure: F.P. Carney, CIBA Vision (E); W.L. Nash, CIBA Vision (E); K.B. Sentell, CIBA Vision (E)

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Overview of the Characteristics of Commercial Lenses Used in the Study

Lenses	Commercial Name	Surface Modification	H2O Content (%)	Monomers	FDA Classification	Manufacturer*
Balafilcon A	PureVision	Plasma oxidation	36%	NVP + TPVC + NCVE + PBVC	Group III	Bausch & Lomb
Galyfilcon A	Acuvue Advance	PVP in reaction mixture	47%	mPDMS + DMA + EGDMA + HEMA + Siloxane macromer + PVP	Group I	Johnson & Johnson, Vision Care
Senofilcon A	Acuvue Oasys	PVP in reaction mixture	38%	mPDMS + DMA + HEMA + Siloxane macromer + TEGDMA + PVF	Group I	Johnson & Johnson, Vision Care
Lotrafilcon A	Night & Day	Plasma polymerization	24%	DMA + TRIS + Siloxane macromer	Group I	CIBA Vision
Lotrafilcon B	O2Optix	Plasma polymerization	33%	DMA + TRIS + Siloxane macromer	Group I	CIBA Vision
Etafilcon A	Acuvue2	None	58%	HEMA + MA	Group IV	Johnson & Johnson, Vision Care

 NVP, N-vinylpyrrolidone; TPVC, tris-(trimethylsiloxysilyl) propylvinyl carbamate; NCVE, N-carboxyvinyl ester; PBVC, poly(dimethylsiloxy) di(silybutanol) bis (Vinyl) carbamate; mPDMS, monofunctional polydimethylsiloxane; DMA, N,N-dimethylacrylamide; EGDMA, ethyleneglycol dimethacrylate; HEMA, poly-2-hydroxyethylmethacrylate; PVP, polyvinyl pyrrolidone; TEGDMA, tetraethylenglycol dimethacrylate; MA, methacrylic acid; FDA, Food and Drug Administration.

*  Bausch & Lomb, Rochester, NY; Johnson & Johnson Vision Care, Jacksonville, FL; CIBA Vision, Duluth, GA.

Figure 1.

 

The adsorption of CH by various silicone hydrogel lenses over 20 days of exposure to concentrations comparable to tear film concentrations. Each point represents a population of 10 lenses.

Figure 2.

 

The adsorption of PE by various silicone hydrogel lenses over 20 days of exposure to concentrations comparable to tear film concentrations. Each point represents a population of 10 lenses. Note the difference in scale between Figures 1 and 2 .