Miriam Heynen

Ex Vivo Protein Deposition on Bi-Weekly Silicone Hydrogel Contact Lenses

Purpose. This study investigated the protein deposition that occurs on daily wear silicone hydrogel (SH) lenses, after 2 weeks of wear. Methods. A total of 40 subjects were divided into equal groups, based on their habitual SH contact lens [CIBA Vision O2OPTIX (O2); Johnson & Johnson ACUVUE ADVANCE with HYDRACLEAR (ADV); Bausch & Lomb PureVision (PV); CIBA Vision Night & Day (ND)]. A randomized, double-masked, cross-over study was conducted in which subjects wore either their habitual SH material or Johnson & Johnson ACUVUE OASYS with HYDRACLEAR PLUS (OAS) for 2 weeks. At the end of the 2-week period, lenses were collected for analysis of total protein, total lysozyme, and percent denatured lysozyme. Results. Total protein was greatest for PV (33 ± 6 &mgr;g/lens), with other lenses depositing <10 &mgr;g/lens. The PV amount was greater than other materials (p < 0.001), with no significant differences between the other three materials (p > 0.05). Total lysozyme was also greatest for the PV lens (11 ± 3 &mgr;g/lens), with other lenses depositing <2 &mgr;g/lens. The PV amount was greater than all other materials (p < 0.001). ADV and OAS were greater than O2 (p < 0.01) but were not different from each other (p > 0.05). The percentage of lysozyme that was denatured was greatest for ND (90 ± 8%) and lowest for PV (23 ± 10%). The lysozyme extracted from ND and O2 lenses was significantly more denatured than that extracted from the other lens materials (p < 0.001). There were no significant differences for lysozyme activity between ND and O2 (p > 0.05) or between ADV, OAS, and PV (p > 0.05). The amount of denatured lysozyme/lens was <3 &mgr;g/lens for all materials. Lysozyme as a percentage of the total protein deposited ranged from 32 (PV) to 6% (O2). Conclusions. This study confirms that all SH lenses deposit low levels of protein, and that the amount and percentage of denatured lysozyme can vary, depending on the overall surface charge of the material and absence or type of surface treatment.

The Impact of Tear Film Components on In Vitro Lipid Uptake

Purpose. To analyze the influence of various tear film components on in vitro deposition of two lipids (cholesterol and phosphatidylcholine) on three contact lens materials. Methods. Etafilcon A, balafilcon A, and senofilcon A were incubated in four different incubation solutions for 3 or 14 days: an artificial tear solution containing lipids and proteins, a protein tear solution containing proteins and the lipid of interest, a lipid tear solution containing lipids and no proteins, and a single lipid tear solution containing the lipid of interest only. Each incubation solution contained one of the two radiolabeled lipids: 14C-cholesterol (C) or 14C-phosphatidylcholine (PC). After soaking, lenses were removed from the incubation solution, the lipids were extracted and quantified using a beta counter, and masses of lipid were calculated using standard calibration curves. Results. This experiment examined several different parameters influencing lipid deposition on contact lenses, including lens material, length of incubation, and the composition of the incubation solution. Overall, lipid deposited differently on different lens materials (p < 0.0005), with the order of deposition most commonly being balafilcon > senofilcon > etafilcon. Incubation solution had a large impact on how much lipid was deposited (p < 0.00001), although cholesterol and phosphatidylcholine demonstrated different deposition patterns. Lipid deposition after 14 days of incubation was consistently greater than after 3 days (p < 0.02). Conclusions. This in vitro study demonstrates that C and PC deposition are cumulative over time and that silicone hydrogel materials deposit more lipid than group IV conventional hydrogel materials. It also clearly demonstrates that deposition of C and PC is influenced by the composition of the incubation solution and that in vitro models must use more physiologically relevant incubation solutions that mimic the natural tear film if in vitro data is to be extrapolated to the in vivo situation.

Quantification of non-polar lipid deposits on senofilcon a contact lenses.

Purpose. To quantify non-polar lipids deposited on senofilcon A silicone hydrogel contact lenses (J&J Acuvue OASYS) when disinfected with a no-rub one-step hydrogen peroxide system (CIBA Vision ClearCare) and a care system preserved with Polyquad & Aldox (Alcon OPTI-FREE RepleniSH). Methods. Thirty existing soft lens wearers symptomatic of dryness were enrolled into a 4-week prospective, randomized, bilateral eye (lens type), cross-over (care regimen), daily wear, double masked study. Subjects were refitted with senofilcon A lenses, which were replaced biweekly. During each period of wear, participants used either the peroxide or preserved system. After each period of wear, lenses were collected and lipid was extracted using 1.5 ml of a 2:1 chloroform:methanol solution for 3 h at 37°C. Lens extracts were analyzed for non-polar lipids [cholesterol oleate (CO), cholesterol, oleic acid (OA), triolein, and OA methyl ester] using normal phase high-performance liquid chromatography. Results. The total lipid (sum of CO and cholesterol) detected was 34 ± 28 &mgr;g/lens for the peroxide-based system and 22 ± 21 &mgr;g/lens for the system preserved with Polyquad and Aldox (p = 0.029). Although there was no difference between products for cholesterol (1.4 vs. 1.3 &mgr;g/lens; p = 0.50), use of a system preserved with Polyquad and Aldox resulted in significantly less deposited CO (33 ± 28 vs. 21 ± 20 &mgr;g/lens; p = 0.033). Approximately, 95% of the detectable lipid deposited on the material was CO, followed by cholesterol. OA and triolein contributed <1% of the total lipid and no OA methyl ester was found on any of the lenses. Conclusions. A care system preserved with Polyquad and Aldox removed higher amounts of CO from senofilcon A contact lenses used for 2 weeks than a peroxide-based system, in soft lens wearers who were symptomatic of dry eye.

Impact of Tear Film Components on Lysozyme Deposition to Contact Lenses

Purpose. To investigate the impact of lactoferrin and lipids on the kinetic deposition of lysozyme on silicone and conventional hydrogel lenses, using a complex artificial tear solution (ATS). Methods. Two silicone hydrogel lenses (AIR OPTIX AQUA; lotrafilcon B and ACUVUE OASYS; senofilcon A) and two conventional hydrogel lenses (ACUVUE 2; etafilcon A and PROCLEAR; omafilcon A) were investigated. Lenses were incubated in four different solutions: a complex ATS consisting of various salts, lipids, proteins, and mucins, an ATS without lactoferrin (ATS w/o Lac), an ATS without lipids (ATS w/o Lip), and an ATS without lactoferrin and lipids (ATS w/o Lac & Lip), each containing 2% radiolabeled (125I) lysozyme (1.9 mg/ml). After each time point (4, 12 h and 1, 2, 3, 5, 7, 14, 21, 28 days), the amount of lysozyme per lens was quantified. Results. After 28 days, lotrafilcon B lenses incubated in ATS deposited significantly less lysozyme (9.7 ± 1.4 &mgr;g) than when incubated in solutions not containing lactoferrin and lipids (more than 11.8 &mgr;g) (p < 0.001). Lysozyme uptake to senofilcon A lenses was higher in ATS w/o Lip (5.3 ± 0.1 &mgr;g) compared with other solutions (less than 3.9 &mgr;g) (p < 0.001). Etafilcon A lenses deposited the most lysozyme in all four solutions compared with the rest of the lens types (p < 0.001). For etafilcon A lenses, less lysozyme was deposited when incubated in ATS w/o Lip (588.6 ± 0.4 &mgr;g) compared with the other solutions (more than 642.6 &mgr;g) (p < 0.001). Omafilcon A lenses in ATS w/o Lac accumulated significantly less lysozyme (12.8 ± 1.0 &mgr;g) compared with the other solutions (more than 14.2 &mgr;g) (p < 0.001). Conclusions. An ATS containing lactoferrin and lipids impacts lysozyme deposition on both silicone and conventional hydrogel contact lenses. When performing in vitro experiments to study protein deposition on contact lenses, more complex models should be used to better mimic the human tear film.

The impact of intermittent air exposure on lipid deposition.

Purpose. To analyze the impact of intermittent air exposure on the in vitro deposition of two radioactive lipids on various contact lens (CL) materials, using a custom-designed model blink cell. Methods. Six different CL materials (balafilcon A, lotrafilcon B, comfilcon A, senofilcon A, etafilcon A, and omafilcon A) were mounted on the model blink cell pistons, which cycled the lenses in and out of a complex artificial tear solution (ATS) that contained a trace amount of 14C-cholesterol or 14C-phosphatidylcholine. For the short-term experiment, air-exposed lenses were continuously cycled in and out of the ATS for 10 h. Longer term incubations for 6 days were tested with lotrafilcon B and balafilcon A materials incubated in 14C-cholesterol ATS. The air-exposed CLs were cycled for 14 h then submerged for 10 h each day. For both experiments, the control lenses were submerged for the entire test period. After incubation, lenses were processed, and deposited masses were quantified. Results. Exposure to air resulted in increased amounts of cholesterol deposited by 1.6 to 4.3 fold on omafilcon A, balafilcon A, comfilcon A, and senofilcon A (p ⩽ 0.03) compared with submerged lenses. No differences in deposition were observed for etafilcon A and lotrafilcon B (p > 0.05). The longer term incubation of lotrafilcon B and balafilcon A showed statistically significant increases in cholesterol deposition for both air-exposed lens materials (p < 0.02) with the increase in deposition 1.8× and 2.8×, respectively. For phosphatidylcholine, all air-exposed lenses had increased masses of deposition. These deposits were statistically greater by 1.1 to 1.6 times for omafilcon A, comfilcon A, lotrafilcon B, and senofilcon A (p < 0.04), but not statistically different for etafilcon A or balafilcon A (p > 0.05). Conclusions. This study found that lipid deposition profiles are CL material dependent and that intermittent air exposure can influence the mass of lipid deposited.

Release of Ciprofloxacin-HCl and Dexamethasone Phosphate by Hyaluronic Acid Containing Silicone Polymers

The purpose of this study was to determine the effect of the covalent incorporation of hyaluronic acid (HA) into conventional hydrogel and hydrogels containing silicone as models for contact lens materials on the uptake and release of the fluoroquinolone antibiotic ciprofloxacin and the anti-inflammatory steroid dexamethasone phosphate. A 3 mg/mL ciprofloxacin solution (0.3% w/v) and a 1 mg/mL dexamethasone phosphate solution (0.1%) was prepared in borate buffered saline. Three hydrogel material samples (pHEMA; pHEMA TRIS; DMAA TRIS) were prepared with and without the covalent incorporation of HA of molecular weight (MW) 35 or 132 kDa. Hydrogel discs were punched from a sheet of material with a uniform diameter of 5 mm. Uptake kinetics were evaluated at room temperature by soaking the discs for 24 h. Release kinetics were evaluated by placing the drug-loaded discs in saline at 34 °C in a shaking water bath. At various time points over 6–7 days, aliquots of the release medium were assayed for drug amounts. The majority of the materials tested released sufficient drug to be clinically relevant in an ophthalmic application, reaching desired concentrations for antibiotic or anti-inflammatory activity in solution. Overall, the silicone-based hydrogels (pHEMA TRIS and DMAA TRIS), released lower amounts of drug than the conventional pHEMA material (p < 0.001). Materials with HA MW132 released more ciprofloxacin compared to materials with HA MW35 and lenses without HA (p < 0.02). Some HA-based materials were still releasing the drug after 6 days.

Factors that influence in vitro cholesterol deposition on contact lenses.

Purpose The purpose of this study was to analyze the impact that incubation time, lipid concentration, and solution replenishment have on silicone hydrogel (SiHy) and conventional hydrogel (CH) contact lens cholesterol deposition via in vitro radiochemical experiments. Methods Four SiHy (senofilcon A, lotrafilcon B, comfilcon A, balafilcon A) and two CH (etafilcon A and omafilcon A) contact lenses were incubated in an artificial tear solution (ATS) that contained major tear film proteins, lipids, salts, salts, and a trace amount of radioactive 14C-cholesterol. Lenses were incubated for various incubation times (1, 3, 7, 14, or 28 days), with three concentrations of lipid (0.5×, 1×, 2× tear film concentration) and with or without solution replenishment to assess each variable’s impact on cholesterol deposition. After incubation, the lenses were extracted using 2:1 chloroform:methanol, extracts were analyzed in a beta counter and masses (micrograms per lens) were extrapolated from standard curves. Results Within the SiHy materials, balafilcon A deposited the greatest amount of cholesterol (p < 0.001) and lotrafilcon B the lowest (p < 0.001). The CH lens materials showed significantly lower uptake amounts than any of the SiHy lens materials (p < 0.001). The uptake of cholesterol ranged from 0.01 ± 0.01 &mgr;g/lens to 3.22 ± 0.34 &mgr;g/lens for all lens materials. Kinetic uptake of cholesterol was shown to be continuous throughout the 28 days of incubation without plateau (p < 0.001), and varying the lipid concentration did impact the resulting cholesterol deposition (p < 0.001). Replenishing the ATS every other day also affected cholesterol deposition throughout the experiment. Overall, the deposition pattern was 2× > replenishing > 1× > 0.5×. Conclusions Overall, SiHy lenses deposit significantly more cholesterol than CH lens materials, and the mass of lipid deposited is dependent on the contact lens material, length of incubation, concentration of lipids in the ATS, and the replenishment of ATS.

Impact of tear film components on the conformational state of lysozyme deposited on contact lenses

PURPOSE To investigate the impact of lactoferrin and lipids on the kinetic denaturation of lysozyme deposited on silicone and conventional hydrogel lenses, using a complex artificial tear solution (ATS). METHODS Two silicone hydrogel lenses (AIR OPTIX AQUA; lotrafilcon B and ACUVUE OASYS; senofilcon A) and two conventional hydrogel lenses (ACUVUE 2; etafilcon A and PROCLEAR; omafilcon A) were incubated in four solutions: an ATS, ATS without lactoferrin, ATS without lipids, and ATS without lactoferrin and lipids. At various time points over a 28-day period, the percentage of active lysozyme per lens was determined using a fluorescence activity assay and an ELISA. RESULTS After 28 days, the percentage of active lysozyme extracted from etafilcon A lenses in all solutions was significantly higher than all other lens materials (p < 0.001). For lotrafilcon B, senofilcon A, and omafilcon A lenses, lysozyme denaturation was greatest during the first week of incubation and before reaching a plateau (p > 0.05). The inclusion of lipids in the ATS significantly increased the lysozyme denaturation on both silicone hydrogel materials (p < 0.001), while in the presence of lactoferrin, lysozyme activity on senofilcon A lenses was significantly higher (p < 0.001). Lysozyme activity on both conventional lenses was not significantly affected by either lactoferrin or lipids (p > 0.05). CONCLUSIONS Lactoferrin and lipids have an impact on the denaturation of lysozyme deposited onto silicone hydrogel contact lenses, while conventional hydrogel lenses were unaffected. Future in vitro studies should consider the impact of tear film components when investigating protein deposition and denaturation on contact lenses.

Optimization of a Fluorescence-based Lysozyme Activity Assay for Contact Lens Studies

ABSTRACT Purpose: To optimize a fluorescence-based lysozyme activity assay to investigate the conformational state of lysozyme in solution and to determine the impact of extraction and evaporation procedures and the possible interference of contact lens materials on lysozyme activity. Methods: The fluorescence-based lysozyme activity assay, Enzchek (Molecular Probes Inc, Eugene, OR) which utilizes fluorescently quenched Micrococcus lysodeikticus, was compared to the gold standard, classical lysozyme turbidity assay, using four differently concentrated lysozyme samples (20, 10, 5.0 and 2.0 ng/µL). Furthermore, six differently concentrated lysozyme samples (2.0, 1.0, 0.5, 0.25, 0.125 and 0.01 µg/µL) were quantified using the fluorescence-based assay in the presence of extraction solvents consisting of 0.2% and 0.02% trifluroacetic acid/acetonitrile and following evaporation procedures. Results: A standard curve was generated by the fluorescence-based assay ranging from 2 to 150 ng. The total active lysozyme quantified in the four lysozyme samples was not significantly different between the two assays (p > 0.05) and the concordance correlation coefficient was determined to be 0.995. However an average discrepancy between the two assays was found to be 0.474 ng, with the turbidity assay typically reporting higher active lysozyme measurements. The sensitivity of the fluorescence-based assay was higher than the classical turbidity assay when quantifying 20 ng or less active lysozyme. Following the extraction and evaporation procedures and the addition of lens extracts, the total active lysozyme recovered was 95% or greater. Conclusions: In comparison to the classical turbidity assay, the fluorescence-based assay is a very sensitive method, making it a favorable technique, particularly when studying contact lens materials that deposit relatively low levels of lysozyme.

Using an In Vitro Model of Lipid Deposition to Assess the Efficiency of Hydrogen Peroxide Solutions to Remove Lipid from Various Contact Lens Materials

Purpose: To test the ability of two commercially available hydrogen peroxide disinfection solutions, one containing a surfactant and one without, to remove lipid from various contact lens materials using in vitro radiochemical experiments. Methods: Etafilcon A, senofilcon A and balafilcon A contact lens materials were incubated in an artificial tear solution (ATS) containing a mixture of lipids, proteins, mucin and either 14C-cholesterol or 14C-phosphatidylcholine for 8 h. Following incubation, the lenses were removed, rinsed, and placed for 16 h in either a surfactant-containing peroxide solution (ClearCare®), a peroxide solution devoid of a surfactant (AOSept®) or stored without solution (control). This process was repeated every day for 1 week. The lenses were extracted with a previously optimized extraction protocol, evaporated, re-suspended, fluor added and counted for their radioactive signals. Masses of lipids deposited were calculated based on standard calibration curves, the disinfection solutions were compared and repeated measures ANOVA and post hoc statistical analysis was completed using Statistica 9. Results: The results of this experiment found that daily disinfection with hydrogen peroxide solutions reduced the amount of cholesterol and phosphatidylcholine deposited on the three contact lens materials examined, however in many cases the reduction in deposition was less than 15% when compared to the control. Disinfection with the solution containing the surfactant (ClearCare), resulted in the least deposited cholesterol and phosphatidylcholine for all materials, however not all of the comparisons were statistically significant. Conclusions: Overall, ClearCare hydrogen peroxide disinfection solution containing Pluronic 17R4 removed the most lipid from lenses when compared to the non-surfactant containing AOSept or the control, for both lipids and all lens materials. However, the differences found were quite small at times and whether these differences are clinically significant are yet to be determined.