Protein deposition on contact lens materials is a complex process and is dependent on several factors, including chemical composition of the lens material, material water content, and surface charge.1–9 In addition to the aforementioned factors, studies have shown that the fabrication process and the surface regularity of the lens material can also influence the deposition of tear components on to lens materials.10–14 Previous in vitro and ex vivo studies have shown that proteins increasingly accumulate on lens materials over time, with longer incubation times or in-eye wearing periods resulting in increased deposition.5,6,8,10–12
A recent study that investigated the proteomic profile of contact lens deposition on silicone hydrogel (SiHy) lens materials using mass spectrometry identified lysozyme, lipocalin, lactoferrin, lacritin, proline-rich 4 and Ig alpha as frequently recognized tear proteins.15 Among the unique tear proteins that are found on hydrogel lens materials, lysozyme is a bacteriolytic enzyme with a relatively small molecular weight (14 kDa) and a positive charge at neutral pH. Once lysozyme firmly adsorbs onto contact lens materials, it tends to denature fairly rapidly,3,16–18 which might potentially result in a variety of immunological responses, including contact lens–associated papillary conjunctivitis.19–22
Currently available SiHy lens materials are cast molded and are available only in a limited range of parameters, which limit their usage. A novel SiHy lens material has been developed by CIBA Vision (sifilcon A), which is lathe cut, allowing for an increased parameter range to be fitted, and for practitioners to “custom-design” the lens in terms of diameter, base curve, and back vertex power.23,24 This lens is also unique in that it is currently the only SiHy lens to be replaced on a 3-month basis, which is considerably longer than other currently available SiHy lens materials, which are replaced every 2 or 4 weeks. The use of such a lens has been welcomed by contact lens practitioners, as it opens up the opportunity to fit SiHy lenses to patients with parameters that are typically outside those currently available.25,26
Several ex vivo studies, which have investigated protein deposition on commercially available cast-molded SiHy lens materials after 2 or 4 weeks of wear, have shown that SiHy lenses deposit significantly lower quantities of protein than conventional FDA group II or IV lens materials.16,17,27–31 Similar results were obtained through in vitro studies, which investigated the protein deposition on contact lenses by artificially “soiling” various lens materials with tear proteins for time periods ranging from 1 h to 1 month.18,32–36 A previous ex vivo study that examined the protein deposition on SiHy lens materials after 2 weeks of wear has shown that lotrafilcon-, galyfilcon-, and senofilcon-based lens materials deposit 5 to 7 μg of total protein per lens, whereas balafilcon lens materials deposit 27 μg of total protein per lens.29 However, to date, no study has investigated the protein deposition on a lathe-cut SiHy lens material, particularly one used for 3 months of wear. Moreover, no study has investigated the conformational state of the deposited protein on any contact lens material after 3 months of lens wear. Thus, the purpose of this study was to determine the quantity of total protein, total lysozyme, and the conformational state of lysozyme deposited on a novel, lathe-cut SiHy lens material after 3 months of in-eye wear.
MATERIALS AND METHODS
Study Design and Collection of Worn Contact Lenses
Ethics clearance was obtained from the Office of Research Ethics at the University of Waterloo before commencement of the study. The study was performed in accordance with the tenets of the Declaration of Helsinki. Informed consent was obtained from all participants before enrolment in the study. This study was conducted as a prospective, bilateral, daily-wear, 9-month clinical evaluation at the Centre for Contact Lens Research, School of Optometry, University of Waterloo.
All the participants were adapted daily-wear hydrogel soft lens wearers. A total of 26 participants (18 women and 8 men; mean age, 31.6 ± 10.75 yr; range, 18 to 54 yr) were enrolled, of which two participants discontinued from the study. Of these, one discontinued because of poor visual quality and the other discontinued because of discomfort and dryness with the lenses. The sifilcon A lens materials were ordered for the participants based on the screening examination and trial lens fitting, and were fitted according to the manufacturer's guidelines.23 Lenses were worn for three consecutive 3-month periods, with lenses being replaced after each period of wear. The commercially available multipurpose solution CLEAR CARE (CIBA Vision, Duluth, GA) was used during this study. On removal at the end of each daily-wear period, participants were instructed to rub the lenses for 5 s with the CLEAR CARE solution, before soaking in fresh solution overnight. Participants were advised to exercise special caution when using the peroxide-based care system with the rubbing step, to avoid getting any peroxide in their eye. They were advised to remove both lenses and place them in the open “baskets” of the lens case before they began the rub/rinse step with either lens, to avoid removing the second lens with a peroxide-laden finger. No enzyme removal systems or stand-alone surfactant cleaners were allowed. Participants habitually using rewetting drops were permitted to continue to use these drops as required.
The clinical results from this study are the subject of another article and will not be described here. Lenses collected from participants were only collected from those who were successful with the new lens type.
Collection of Worn Contact Lenses
On completion of 3 months of daily wear, lenses from the left eye were collected (using non-powdered surgical gloves) and placed in individual, sealed glass vials containing 1.5 mL of a 50:50 mix of 0.2% trifluoroacetic acid and acetonitrile (ACN/TFA), as described previously.17,28,37,38 The vials were incubated in the dark at room temperature for 24 h after which the aliquots of lens extracts were transferred to sterile Axygen microcentrifuge tubes and evaporated to dryness in a Savant Speed Vac (Halbrook, NY). Dried protein pellets were stored at −80°C for up to 2 weeks before reconstitution.
Reagents and Materials
Sifilcon A lenses (O2OPTIX Custom) were provided by CIBA Vision. The known properties of this material are given in Table 1. The PhastSystem components were described in detail previously.17 Immunoblot polyvinylidene difluoride membranes and protein assay reagents were purchased from Bio-Rad Laboratories (Mississauga, ON, Canada). Polyclonal rabbit anti-human lysozyme was purchased from Cedarlane Laboratories (Hornby, ON, Canada) and goat anti-rabbit IgG-horseradish peroxidase was purchased from Sigma (St. Louis, MO). Human lysozyme (neutrophil) and BioStab Biomolecule Storage Solution (Sigma # 92,889) were purchased from VWR (Toronto, ON, Canada). Micrococcus lysodeikticus and all other reagents purchased were of analytical grade and obtained from Sigma Aldrich.
Measurement of Total Lysozyme Deposition—Electrophoresis and Immunoblotting
Lenses collected in ACN/TFA were incubated in the dark at room temperature for 24 h and the samples were processed as described previously.28,38 Lysozyme standards were prepared fresh on the day of analysis from a 1.0 μg/μL frozen stock of purified human neutrophil lysozyme with modified reconstitution buffer, pH 8.0 and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by Western blotting to polyvinylidene difluoride membranes. The entire procedure is described in detail elsewhere.17,28,38
Negative Control—Extraction and Western Blot Analysis of Unworn Lenses
Three new unworn sifilcon A lenses were extracted in ACN/TFA solution and were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting, as described above.
Measurement of Lysozyme Activity
The contact lens extracts were assayed for lysozyme activity using a fresh suspension of M. lysodeikticus for each sample, as described previously.17,18,28Micrococcal cells were suspended in 50 mM sodium phosphate buffer (pH 6.3) to an initial optical density of 1.0 at 450 nm (Multiskan Spectrum ELISA Plate Reader, fitted with a micro-cuvette, ThermoLabsystems). Human neutrophil lysozyme standard (2.5, 5, 12.5, 50, 150, and 250 ng) was run concurrently with the samples. The mass of active lysozyme in contact lens extracts was extrapolated from the native lysozyme standard curve, as described previously.17,18,28
Measurement of Total Protein Deposition
A Bradford assay39,40 was used to determine the total protein deposited on the sifilcon A lens material; however, modifications were made to quantify the contact lens extracts. Bovine serum albumin (0, 0.5, 1, 2, 4, 6, 8, and 10 μg) was used as the protein standard. All tubes were neutralized with 25 μL 0.5 M phosphate pH 7.2 and 145 μL water, and neutrality was confirmed with pH paper. The total volume for this initial sample preparation was 190 μL (20 μL + 25 μL + 145 μL water). Calcium chloride (10 μL, 500 mM) was added and all tubes were mixed well by vortexing. The samples were allowed to precipitate for 5 min and 1 mL of 99.9% ethanol was added. The tube was vortexed and centrifuged (15,000g for 1 min) and the supernatant was aspirated, avoiding the pellet. One milliliter of 90% (vol/vol) ethanol was then added which was followed by vortexing, centrifuging, and aspiration. The samples were then dried in the SpeedVac for 15 min. Fifty microliter of the Bradford reagent was added to the samples and the samples were placed statically for 10 min at room temperature. The tubes were then placed in boiling water for 30 s to dissolve the precipitates, followed by vortexing. Once the tubes returned to room temperature, they were centrifuged briefly, such that the Bradford reagent sedimented at the bottom of the tube. Two hundred microliter of cold NaCl was added to the samples and were vortexed twice. Two hundred microliter of the sample was then transferred to a 96-well microtiter plate and the absorption ratio was read within 10 min. Samples were read on three occasions and the average of these was recorded. A standard curve was prepared and the line of best fit was determined. The sample A595 values were compared with the curve to calculate the amount of total protein in the samples.
Atomic Force Microscopy
The surface morphology of new unworn sifilcon A lenses was determined using atomic force microscopy (AFM). As a comparison, new unworn lotrafilcon B (O2Optix) lenses were also imaged in an identical fashion (see Table 1 for details). Unworn lenses (n = 3 of each type) were rinsed thoroughly with Millipore deionized water (to remove the packaging solution) and were imaged using an Explorer AFM (Veeco Instruments, Santa Barabara, CA) in tapping mode with MikroMasch NSC15/A1BS tips (r < 10 nm) from SOMTIPS.COM, as described in detail elsewhere.41 All lenses were −10.00 D and representative images from those obtained are reported.
Table 2 shows the total protein, total lysozyme, denatured lysozyme in microgram per lens, and the percentage denatured lysozyme after 3 months of daily wear of the sifilcon A lens material. For comparison purposes, Table 2 also shows the protein deposition data from another ex vivo study from our group,29 which reports on the total protein, total lysozyme, denatured lysozyme in microgram per lens, and the percentage denatured lysozyme after 2 weeks of wear of lotrafilcon A, lotrafilcon B, balafilcon A, galyfilcon A, and senofilcon A lens materials.
No signal was seen on Western blots run on unworn sifilcon A lenses after being subject to the same procedures, confirming that there was no background interference from the lens polymer.
Figs. 1 and 2 show the AFM images of lotrafilcon B and sifilcon A lens materials, respectively, at 20 × 20 μm scan size.
This is the first study to report on protein deposition levels on a novel, custom-made, lathe-cut SiHy lens material after 3 months of daily wear. Traditionally, lathe-cut lens materials undergo a “polishing” step to eliminate any surface irregularities. However, the sifilcon A lens materials do not undergo this polishing step because these lenses undergo a state-of-the-art custom lathing process through the InnoLathe manufacturing technology (CIBA Vision, personal communication), obviating the necessity for this final polishing step. The finished lenses then undergo a similar plasma surface treatment to that seen with CIBA Vision's other SiHy materials, which are permanently modified in a gas plasma reactive chamber using a mixture of trimethylsilane oxygen and methane to create a permanent, ultrathin (25 nm), high refractive index, continuous hydrophilic surface.42–46
The AFM image of the lotrafilcon B lens material (Fig. 1) shows that the surface of these lenses exhibits characteristic multiple linear marks that are similar to that previously reported for lotrafilcon A47,48 and lotrafilcon B lenses.41 Surprisingly, these characteristic linear “lines” are not seen on the surface of the lathe-cut sifilcon A material (Fig. 2), as linear marks are commonly seen on lenses that are fabricated through a lathe-cutting process.49,50 Interestingly, the surface of the sifilcon A lens material demonstrates a porous, “sponge-like” surface topography similar to that of galyfilcon A and senofilcon A lens materials.41 This is of interest, as it seems that lathing a “rubbery,” siloxane-based material, such as sifilcon A, produces a different topography to that typically seen in lathed HEMA materials. It is clear from Figs. 1 and 2 that the surface topography of the lathe-cut sifilcon A material is “less smooth” than that of the lotrafilcon B lens material. It has previously been shown with HEMA-based materials that lathe-cut lenses have surfaces that are generally “rougher” than those fabricated by cast molding.49,51
It is noteworthy to compare the total protein deposited on the sifilcon A lens material with that deposited on a conventional hydrogel contact lens after 3 months of wear. In this study, sifilcon A deposited 5.3 ± 2.3 μg/lens of total protein (Table 2) after 3 months of wear, as compared with a conventional hydrogel FDA group II material (vasurfilcon A), which deposited 106 ± 16 μg/lens of total protein52 after the same period of wear. In the other study, Bausch & Lomb's ReNu Multi-Purpose was used as the care regimen, which was used with a “rub-and-rinse” format. The vasurfilcon A lens material has methyl methacrylate and N-vinyl pyrrolidone as its principal monomers; thus, it is non-ionic, high water content (74%) material, which is classified as a FDA group II material. It has also been shown in an earlier in vitro study using a radiolabeling technique that FDA group II lens materials deposit significantly lower amounts of lysozyme than a high water content, ionic FDA group IV lens material, but deposit higher amounts of lysozyme than most SiHy lens materials.32 Despite the fact that the care regimen and the methods used to examine the total protein were different in these two studies, this information puts into perspective the fact that SiHy lens materials still deposit substantially less protein than conventional lens materials, even after periods of wear longer than that typically seen with most SiHy lenses.
It is also interesting to note that the total protein and total lysozyme deposited on the sifilcon A lens material is similar to that found on similar surface-coated SiHy lens materials (such as lotrafilcon A and lotrafilcon B) after 2 weeks of lens wear.29 The results from the other ex vivo study29 showed that lotrafilcon A, lotrafilcon B, galyfilcon A, and senofilcon A deposited 5.2 ± 2.2, 6.6 ± 3.4, 6.3 ± 3.4, and 4.6 ± 2.5 μg of total protein per lens respectively, whereas balafilcon A deposited 26.9 ± 9.3 μg of total protein per lens after 2 weeks of wear (Table 2). It is also of interest to note that the total protein and total lysozyme deposition on the sifilcon A lens material is similar to that seen on galyfilcon A and senofilcon A lens materials. As mentioned previously, the AFM images from the current study (Fig. 2) show that sifilcon A lens has a similar surface topography to that of galyfilcon A and senofilcon A lens materials.41 Both this study and the other ex vivo study29 used the peroxide-based CLEAR CARE solution as the care regimen. However, in this study the participants used the solution in a rub-and-rinse format, whereas in the other study29 the solution was used only in the “rinse” format. Despite this difference, the levels of deposition were remarkably similar. More work looking at the role of “rubbing-and-rinsing” and its role in keeping the sifilcon A lenses “clean” of deposits is warranted, as this study only investigated protein deposition and not other tear-derived deposits, particularly lipid, which are known to be an issue with SiHy lenses.16,53–55
Previous studies, which have compared the protein uptake by contact lens materials that are fabricated by means of spin-casting, cast-molding, and lathe-cut processes, have shown that lathe-cut lens materials typically attract more deposits than the other fabricating methods.10–12 This enhanced deposition could be due to the presence of an increased number of binding sites on the lens material, which are caused by the fabrication defects on the lathe-cut lenses. A study by Fowler and Gaertner,14 using a scanning electron microscope, also demonstrated that there was “heaping-up” of deposits in the lathe-cut areas of worn contact lenses. In contrast, a study by Kaplan and Gundel13 showed that there was no significant difference in deposition between polished and unpolished lathe-cut lens materials. The results from this study would suggest that factors such as material water content, surface charge, or treatment and material composition play a far bigger role in determining the deposition of tear proteins on hydrogel lenses than the manufacturing method.
This is the first study to determine the conformational state of lysozyme deposited on a SiHy lens material after 3 months of wear. The results from this study show that after 3 months of lens wear, the percentage denatured lysozyme recovered from these materials was 80 ± 10% (Table 2). The percentage denatured lysozyme recovered from these lenses is comparable to that from the lotrafilcon A lens material worn on an extended wear basis for 30 days16 and comparable to lotrafilcon A and lotrafilcon B lens materials after 2 weeks of daily lens wear.29 It is highly unlikely that lysozyme, which is irreversibly bound to the lens polymer, will have retained any biological activity after 3 months of wear. The 20% “active” lysozyme, which is recovered from the sifilcon A lens material, is more likely to be adsorbed “firmly” but not “irreversibly” on to the lens material. In addition, it is speculated that the lysozyme, which has retained its biological activity, might have adsorbed either “reversibly” or “irreversibly” on the lenses on the day when the lens was collected for laboratory analysis.
In conclusion, the results from this study indicate that the extended use of the sifilcon A lens material used in the O2OPTIX Custom SiHy lens is not deleterious in terms of the amount and the quality of protein that is deposited on the lens. These results indicate that even after 3 months of wear, the quantity of protein and the conformational state of lysozyme deposited on these novel lenses is very similar to that found on similar surface-coated SiHy lenses (lotrafilcon A and lotrafilcon B) after 2 to 4 weeks of wear. These results reiterate that SiHy lens materials, although fabricated via a lathe-cutting process, deposit low quantities of total protein and total lysozyme even after extended period of daily wear. Further work is required to determine the location of lysozyme on these lathe-cut lens materials. Lysozyme forms only a portion of the total protein deposited on the sifilcon A lens material; hence, the quantity of other protein types deposited should also be investigated. It would also be of interest to study lipid deposition on these lens materials and also to determine the impact of care regimen on protein/lipid deposition.
The study to examine the clinical performance of the lenses used in this study was funded by CIBA Vision, Duluth, GA. The analytical work, which is the basis of this report, was funded by a Collaborative Research and Development Grant from Natural Sciences and Engineering Research Council of Canada (NSERC) (to LJ). No honoraria, gifts, or other funding was received from the sponsor. One of the authors (LNS) was a recipient of the American Optometric Foundation's William C Ezell Fellowship.
Centre for Contact Lens Research
School of Optometry
University of Waterloo
200 University Avenue West
Waterloo, Ontario N2L 3G1, Canada
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Keywords:© 2009 American Academy of Optometry
contact lens; deposition; lysozyme activity; lathe-cut silicone hydrogel; tear protein