Doerte Luensmann

Protein deposition on contact lenses: The past, the present, and the future

Proteins are a key component in body fluids and adhere to most biomaterials within seconds of their exposure. The tear film consists of more than 400 different proteins, ranging in size from 10 to 2360 kDa, with a net charge of pH 1-11. Protein deposition rates on poly-2-hydroxyethyl methacrylate (pHEMA) and silicone hydrogel soft contact lenses have been determined using a number of ex vivo and in vitro experiments. Ionic, high water pHEMA-based lenses attract the highest amount of tear film protein (1300 μg/lens), due to an electrostatic attraction between the material and positively charged lysozyme. All other types of pHEMA-based lenses deposit typically less than 100 μg/lens. Silicone hydrogel lenses attract less protein than pHEMA-based materials, with <10 μg/lens for non-ionic and up to 34 μg/lens for ionic materials. Despite the low protein rates on silicone hydrogel lenses, the percentage of denatured protein is typically higher than that seen on pHEMA-based lenses. Newer approaches incorporating phosphorylcholine, polyethers or hyaluronic acid into potential contact lens materials result in reduced protein deposition rates compared to current lens materials.

Albumin adsorption to contact lens materials: A review

During contact lens wear, tear film components such as lipids, mucins and proteins tend to deposit on and within the lens material and may cause discomfort, reduced vision and inflammatory reactions. The tear film protein that has attracted most interest when studying contact lens deposition is the small (14 kDa), positively charged protein lysozyme. Albumin, which is a much larger protein (66 kDa) with an overall net negative charge is also of interest, and shows very different adsorption patterns to lysozyme. The concentration of albumin in the tear film is relatively low compared to the concentration in blood serum, but this value increases markedly under various conditions, including when the eye is closed, during contact lens wear and in various dry eye states. Gaining an understanding of the manner in which albumin deposits on biomaterials is of importance for contact lens wear, as well as for other medical applications where HEMA-based materials are used for implants, artificial blood vessels or drug delivery devices. This review paper summarizes the impact of individual material compositions, water content, hydrophobicity and electrostatic attraction on the adsorption behavior of the protein albumin.

Localization of Lysozyme Sorption to Conventional and Silicone Hydrogel Contact Lenses Using Confocal Microscopy

Purpose: To investigate the distribution profile of hen egg lysozyme (HEL) through poly-2-hydroxyethyl methacrylate (pHEMA)-based lens materials and silicone hydrogel (SH) lens materials using confocal laser scanning microscopy (CLSM). Methods: Five silicone SH materials (balafilcon A, lotrafilcon A, lotrafilcon B, galyfilcon A, senofilcon A) and four pHEMA-based materials (alphafilcon A, etafilcon A, omafilcon A, vifilcon A) were incubated in 1.9 mg/ml protein solution for 24 hours. The protein solution consisted of HEL, which was conjugated with either fluorescein isothiocyanate (FITC) or lucifer yellow VS dilithium salt (LY). CLSM (Zeiss LSM 510 META) identified the location of the fluorescently labeled protein by using 1 μ m depth scans through the lens. In a second experiment, lenses were incubated with 2% 125 I labeled HEL to determine the amount of deposited protein on each lens. Both techniques were combined to describe the individual HEL profiles. Results: After the incubation in fluorescently labeled HEL, all pHEMA-based materials and the SH material balafilcon A accumulated protein throughout the entire lens material, while, for the SH lenses lotrafilcon A and lotrafilcon B, HEL was primarily detected on the lens surface alone. Differences in protein uptake pattern due solely to the two conjugated dyes were most apparent for the SH materials galyfilcon A and senofilcon A; HEL was detected throughout these lenses when conjugated with LY but accumulated primarily on the surface when conjugated with FITC. Conclusion: CLSM in combination with a radiolabel technique can describe both the location and degree of protein deposition on different contact lens materials.

Confocal microscopy and albumin penetration into contact lenses.

Purpose. To develop a novel in vitro method to detect the depth of penetration of the tear film protein albumin into contact lens materials using confocal laser scanning microscopy (CLSM). Methods. A poly-HEMA-based hydrogel (etafilcon A) and a silicone hydrogel material (lotrafilcon B) were examined. In vitro, bovine serum albumin (BSA) was labeled with 5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein hydrochloride (DTAF). The lenses were incubated in this protein solution (0.5 mg/ml) at 37°C. After 1 and 7 days incubation, the lenses were examined using CLSM (Zeiss 510, config. META 18) and the location of the fluorescently labeled BSA was identified. Results. BSA adsorption on the surface and penetration into the lens matrix occurred at a higher concentration for etafilcon compared to lotrafilcon (p < 0.001). For both materials, BSA was detected on the surface after 1 day of incubation. Significant levels of BSA were detected within the matrix of etafilcon after as little as 1 day (p < 0.001), but no BSA was detected in the matrix of lotrafilcon at any time (p > 0.05). Conclusion. CLSM can be successfully used to examine the depth of penetration of fluorescently labeled proteins into various hydrogel polymers. Our results show that etafilcon lenses both adsorb BSA on the surface and absorb BSA within the matrix, whereas lotrafilcon B adsorbs small amounts of BSA on the surface only.

The prevalence and impact of high myopia.

Abstract: Myopia has gained increasing interest in recent years, particularly because of its increase in prevalence across populations worldwide. The onset of myopia has shifted to a younger age, and the number of high myopes with prescriptions of more than −5.00 D has increased over the last few decades. High myopia is strongly associated with a greater incidence of pathologic complications, has shown to impact vision-related quality of life in children and adults and is further associated with certain contact lens complications. Different pharmaceutical and optical treatment options are currently under investigation with a common goal to slow down the rate of myopia progression.

Hyaluronic acid as an internal wetting agent in model DMAA/TRIS contact lenses

Model silicone hydrogel contact lenses, comprised of N,N-dimethylacrylamide and methacryloxypropyltris (trimethylsiloxy) silane, were fabricated and hyaluronic acid (HA) was incorporated as an internal wetting agent using a dendrimer-based method. HA and dendrimers were loaded into the silicone hydrogels and cross-linked using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide chemistry. The presence and location of HA in the hydrogels was confirmed using X-ray photoelectron spectroscopy and confocal laser scanning microscopy, respectively. The effects of the presence of HA on the silicone hydrogels on hydrophilicity, swelling behavior, transparency, and lysozyme sorption and denaturation were evaluated. The results showed that HA increased the hydrophilicity and the equilibrium water content of the hydrogels without affecting transparency. HA also significantly decreased the amount of lysozyme sorption (p < 0.002). HA had no effect on lysozyme denaturation in hydrogels containing 0% and 1.7% methacrylic acid (MAA) (by weight) but when the amount of MAA was increased to 5%, the level of lysozyme denaturation was significantly lower compared to control materials. These results suggest that HA has great potential to be used as a wetting agent in silicone hydrogel contact lenses to improve wettability and to decrease lysozyme sorption and denaturation.

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 effects of hyaluronic acid incorporated as a wetting agent on lysozyme denaturation in model contact lens materials.

Conventional and silicone hydrogels as models for contact lenses were prepared to determine the effect of the presence of hyaluronic acid on lysozyme sorption and denaturation. Hyaluronic acid was loaded into poly(2-hydroxyethyl methacrylate) and poly(2-hydroxyethyl methacrylate)/TRIS – methacryloxypropyltris (trimethylsiloxy silane) hydrogels, which served as models for conventional and silicone hydrogel contact lens materials. The hyaluronic acid was cross-linked using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in the presence of dendrimers. Active lysozyme was quantified using a Micrococcus lysodeikticus assay while total lysozyme was determined using 125-I radiolabeled protein. To examine the location of hyaluronic acid in the gels, 6-aminofluorescein labeled hyaluronic acid was incorporated into the gels using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide chemistry and the gels were examined using confocal laser scanning microscopy. Hyaluronic acid incorporation significantly reduced lysozyme sorption in poly(2-hydroxyethyl methacrylate) (p < 0.00001) and poly(2-hydroxyethyl methacrylate)/TRIS – methacryloxypropyltris (trimethylsiloxy silane) (p < 0.001) hydrogels, with the modified materials sorbing only 20% and 16% that of the control, respectively. More importantly, hyaluronic acid also decreased lysozyme denaturation in poly(2-hydroxyethyl methacrylate) (p < 0.005) and poly(2-hydroxyethyl methacrylate)/TRIS – methacryloxypropyltris (trimethylsiloxy silane) (p < 0.02) hydrogels. The confocal laser scanning microscopy results showed that the hyaluronic acid distribution was dependent on both the material type and the molecular weight of hyaluronic acid. This study demonstrates that hyaluronic acid incorporated as a wetting agent has the potential to reduce lysozyme sorption and denaturation in contact lens applications. The distribution of hyaluronic acid within hydrogels appears to affect denaturation, with more surface mobile, lower molecular weight hyaluronic acid being more effective in preventing denaturation.

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.