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The purpose of this study was to observe penetration profiles of lysozyme on a variety of contact lens materials by confocal microscopy, to analyze influential factors that are involved in these penetration curves and to suggest possible mechanisms related to the in-eye clinical performance of these materials. An FITC-lysozyme conjugate was synthesized in-house by amine reaction. Contact lenses were incubated in a lysozyme solution with a final concentration of 1.9 mg/mL for various periods before undergoing microscopic analysis. Optimal parameters for confocal scanning were successfully obtained to acquire desired fluorescence signals on various contact lenses. Measurement units were converted into absolute amounts of lysozyme using lysozyme data from ^(125)I gamma counting studies. A rhodamine labeled dextran solution was applied to distingush the surface of the contact lenses under examination. The data from these studies were then used to calculate the theoretical numbers of layers of adsorbed lysozyme on the lens surface. The results show that there were distinct differences in lysozyme penetration in the twelve hydrogel materials examined. A pure pHEMA lens, with a water content of 38%, deposited lysozyme primarily on the lens surface after 24 hours, with full penetration occurring after 4-weeks of incubation. Three types of non-ionic contact lens materials with water contents > 50% exibited rapid penetration within the lens bulk after 24-hours incubation, with increased deposition within the matrix after 4 weeks. Two ionic, high water content polymers (Acuvue 2 and Focus Monthly) exhibited markedly different penetration profiles, particularly after 24 hours, with very rapid and total penetration in Acuvue 2, as compared with partial penetration in Focus Monthly. Modern silicone hydrogel contact lenses can be nominally divided into first generation, plasma-modified materials and second generation materials which incorporate an internal wetting agent such as polyvinyl pyrrolidone (PVP). These materials exhibited different lysozyme deposition profiles. Lysozyme fully penetrated PureVision after 24 hours, whereas no lysozyme penetration occurred on lenses manufactured from Focus Night & Day or O_2Optix, even after 4 weeks. Lenses manufactured from Acuvue Advance and Acuvue OASYS, two second generation silicone hydrogel lenses, also displayed their own characteristic deposition profile. Acuvue Advance always exhibited a partial penetration of lysozyme within the matrix, even after 4 weeks of doping. Interestingly, Acuvue OASYS showed a similar profile to Focus Night & Day and O_2Optix, with predominantly surface deposition occurring. To confirm possible surface adsorption of lysozyme on surface-coated Focus Night & Day and O_2Optix, a rigid polymethylmethacrylate (PMMA) contact lens was used as a model of surface adsorption. A mounting medium containing rhodamine labeled dextran was scanned to distinguish the lens surface, as it was assumed that no surface penetration of the very high molecular weight dextran would occur. Using this model, it was confirmed that surface adsorption of lysozyme occurred on these plasmacoated lens materials, which is similar to that seen with PMMA. In a further experiment, it was seen that lysozyme sorption on Acuvue OASYS exhibits a penetration profile which is different to that seen in Focus Night & Day and O_2Optix, with lysozyme just penetrating the lens surface. The results from the studies described above demonstrated that in 24 hours lysozyme sorption did not achieve a complete monolayer. However, after 4 weeks multi-layer adsorption occurred, with the more hydrophilic materials depositing the most lysozyme. The quantitative measurement of lysozyme penetration on and into contact lens materials by confocal microscopy combined with ^(125)I labelling offers a valuable tool to discover the potential mechanisms of interactions between protein and polymer materials. This study reveals some important information that may be beneficial to contact lens development and will prove to be valuable in other more broad areas of biomedical research in which polymers and biological fluids come into contact. / Thesis / Master of Applied Science (MASc)

Over 60 proteins have been detected in the tear film and among these lysozyme has attracted the greatest attention. Several techniques for elucidating the identity, quantity and conformation of lysozyme deposited on soft contact lenses have been developed. Lysozyme also deposits on the newly introduced silicone hydrogel (SH) lens materials, but in extremely low levels compared to conventional hydrogel lenses. Hence, a major analytical complication with the study of the SH contact lens materials relates to the minute quantity of deposited lysozyme. The first project of this thesis involved the development of a method whereby lysozyme mass extracted from SH lens materials would be preserved over time and would be compatible with an optimized Western blotting procedure. This methodological development was incorporated into a clinical study (CLENS-100?? and Silicone Hydrogels ? CLASH study) wherein the difference in the degree of total protein, the difference in lysozyme deposition and activity recovered from lotrafilcon A SH lens material when subjects used surfactant containing rewetting drops (CLENS-100??) versus control saline was investigated. The remaining experiments were in vitro experiments wherein the lenses were doped in artificial lysozyme solution containing ¹²⁵I-labeled lysozyme. These experiments were performed to gain insight into the kinetics of lysozyme deposition on SH lens materials and also the efficacy of a reagent in extracting lysozyme from SH lens materials. A protocol was developed whereby the percentage loss of lysozyme mass found on lotrafilcon A SH lenses was reduced from approximately 33% to <1% (p<0. 001), following extraction and resuspension. The results from the CLASH study demonstrated that when subjects used a surfactant containing rewetting drop instead of a control saline drop total protein deposition (1. 2??0. 7 ??g/lens versus 1. 9??0. 8 ??g/lens, p<0. 001), lysozyme deposition (0. 7??0. 5 ??g/lens versus 1. 1??0. 7 ??g/lens, p<0. 001) and percentage lysozyme denaturation (76??10% versus 85??7%, p=0. 002) were all reduced. The results from the kinetics study demonstrated that lysozyme accumulated rapidly on etafilcon A lenses (1 hr, 98??8 ??g/lens), reached a maximum on the 7th day (1386??21 ??g/lens) and then reached a plateau (p=NS). Lysozyme accumulation on FDA Group II and SH lenses continued to increase across all time periods, with no plateau being observed (p<0. 001). The results from the extraction efficiency study showed that 0. 2% trifluoroacetic acid/ acetonitrile was 98. 3??1. 1% and 91. 4??1. 4% efficient in extracting lysozyme deposited on etafilcon A and galyfilcon lenses, while the lysozyme extraction efficiency was 66. 3??5. 3 % and 56. 7??3. 8% for lotrafilcon A and balafilcon lens materials (p<0. 001). The results from these studies re-emphasize that novel SH lens materials are highly resistant to protein deposition and demonstrate high levels of biocompatibility.

The cornea is the anterior most surface of the eye and plays a critical role in vision. A thin fluid layer, the tear film, coats the outer surface of the cornea and serves to protect, nourish, and lubricate the cornea. At the same time, the tear film is responsible for creating a smooth continuous surface, where the majority of refraction takes place in the eye. A significant component of vision quality is determined by the shape of the cornea and stability of the tear film. A dual interferometer system for measuring the dynamic corneal topography is designed, built, verified, and qualified by testing on human subjects. The system consists of two coaligned simultaneous phase-shifting polarization-splitting Twyman-Green interferometers. The primary interferometer measures the surface of the tear film while the secondary interferometer tracks the absolute position of the cornea, which provides enough information to reconstruct the absolute shape of the cornea. The results are high-resolution and high-accuracy surface topography measurements of the in vivo tear film and cornea that are captured at standard camera frame rates. (C) 2016 Society of Photo-Optical Instrumentation Engineers (SPIE)

Introduction Contact lenses suffer from the same problems of deposition that other biomaterials exhibit, being rapidly coated with a variety of proteins, lipids and mucins. The first event observed at the interface between a contact lens and tear fluid is protein adsorption. Protein deposits on contact lenses are associated with diminished visual acuity, dryness and discomfort and lid-related inflammatory changes. The aim of this thesis was to determine the quantity and the conformational state of lysozyme deposited on contact lens materials over various time periods and also to determine the clinical relevance of protein deposits on contact lenses. The specific aims of each chapter of this thesis were as follows: • Chapter 4: To determine the total lysozyme deposition on conventional and silicone hydrogel contact lens materials as a function of time by artificially doping lenses with 125I-labeled lysozyme. • Chapter 5: To determine the conformational state of lysozyme deposited on conventional and silicone hydrogel contact lens materials as a function of time using an in vitro model. • Chapter 6: To quantify the total protein, total lysozyme and the conformational state of lysozyme deposited on a novel, lathe-cut silicone hydrogel contact lens material after three-months of wear. • Chapter 7: To determine the relationship between protein deposition and clinical signs & symptoms after one-day wear of etafilcon lenses in a group of symptomatic and asymptomatic lens wearers. • Chapter 8: To determine the influence of individual tear proteins (lysozyme, lactoferrin and albumin) on the adhesion of Gram positive and Gram negative bacteria to conventional and silicone hydrogel contact lens materials. Methods • Chapter 4: Conventional hydrogel FDA group I (polymacon), group II (alphafilcon A and omafilcon A), group IV (etafilcon A and vifilcon A), polymethyl methacrylate and silicone hydrogel lens materials (lotrafilcon A, lotrafilcon B, balafilcon A, galyfilcon A and senofilcon A) were incubated in a lysozyme solution containing 125I-labeled lysozyme for time periods ranging from 1 hour to 28 days. After each time period, lysozyme deposited on contact lens materials was determined using a Gamma Counter. • Chapter 5: Conventional hydrogel FDA groups I, II, IV and silicone hydrogel lens materials were incubated in lysozyme solution for time periods ranging from 1 hour to 28 days. After each time period, the lysozyme deposited on the lenses was extracted and the sample extracts were assessed for lysozyme activity and total lysozyme. • Chapter 6: 24 subjects completed a prospective, bilateral, daily-wear, nine month clinical evaluation in which the subjects were fitted with a novel, custom-made, lathe-cut silicone hydrogel lens material (sifilcon A). After 3 months of wear, the lenses were collected and total protein, total lysozyme and active lysozyme deposition were assessed. • Chapter 7: 30 adapted soft contact lens wearers (16 symptomatic and 14 asymptomatic) were fitted with etafilcon lenses. Objective measures and subjective symptoms were assessed at baseline and after hours 2, 4, 6 and 8. After 2, 4, 6 and 8 hour time points, lenses were collected and total protein, total lysozyme and active lysozyme deposition were assessed. • Chapter 8: Three silicone hydrogel (balafilcon A, lotrafilcon B & senofilcon A) and one conventional hydrogel (etafilcon A) lens materials were coated with lysozyme, lactoferrin and albumin. Uncoated and protein-coated contact lens samples were incubated in a bacterial suspension of Staphylococcus aureus 31 and two strains of Pseudomonas aeruginosa (6294 & 6206). The total counts and the viable counts of the adhered bacteria were assayed. Results • Chapter 4: Lysozyme accumulated rapidly on conventional hydrogel FDA group IV lenses, reached a maximum on day 7 and then plateaued with no further increase. PMMA showed a deposition pattern similar to that seen on lotrafilcon A and lotrafilcon B silicone hydrogel lenses. After 28 days, conventional hydrogel FDA group IV lenses deposited the most lysozyme. • Chapter 5: After 28 days, lysozyme deposited on group IV lenses exhibited the greatest activity. Lysozyme deposited on polymacon, lotrafilcon A and lotrafilcon B exhibited the lowest activity. Lysozyme deposited on omafilcon, galyfilcon, senofilcon, and balafilcon exhibited intermediate activity. • Chapter 6: The total protein recovered from the custom-made lenses was 5.3±2.3 µg/lens and the total lysozyme was 2.4±1.2 µg/lens. The denatured lysozyme found on the lenses was 1.9±1.0 µg/lens and the percentage of lysozyme denatured was 80±10%. • Chapter 7: Correlations between subjective symptoms and protein deposition showed poor correlations for total protein/ lysozyme and any subjective factor, and only weak correlations between dryness and active lysozyme. However, stronger correlations were found between active lysozyme and subjective comfort. • Chapter 8: Different tear proteins had varying effects on the adhesion of bacteria to contact lens materials. Lysozyme deposits on contact lenses increased the adhesion of Gram positive Staphyloccocus aureus 31 strain, while albumin deposits increased the adhesion of both the Gram positive Staphyloccocus aureus and Gram negative Pseudomonas aeruginosa 6206 & 6294 strains. Lactoferrin deposits increased the total counts of both the Gram positive and Gram negative strains, while they reduce the viable counts of the Gram negative strains. Conclusions • Chapter 4: Lysozyme deposition is driven by both the bulk chemistry and also the surface properties of conventional and silicone hydrogel contact lens materials. The surface modification processes or surface-active monomers on silicone hydrogel lens materials also play a significant role in lysozyme deposition. • Chapter 5: The reduction in the activity of lysozyme deposited on contact lens materials is time dependent and the rate of reduction varies between lens materials. This variation in activity recovered from lenses could be due to the differences in surface/ bulk material properties or the location of lysozyme on these lenses. • Chapter 6: Even after three-months of wear, the quantity of protein and the conformational state of lysozyme deposited on these novel lens materials was very similar to that found on similar surface-coated silicone hydrogel lenses after two to four weeks of wear. These results indicate that extended use of the sifilcon A material is not deleterious in terms of the quantity and quality of protein deposited on the lens. • Chapter 7: In addition to investigating the total protein deposited on contact lenses, it is of significant clinical relevance to determine the conformational state of the deposited protein. • Chapter 8: Uncoated silicone hydrogel lens materials bind more Gram positive and Gram negative bacteria than uncoated conventional hydrogel lens materials. Lysozyme deposited on contact lens materials does not possess antibacterial activity against all bacterial strains tested, while lactoferrin possess an antibacterial effect against certain Gram negative strains tested in this study. This thesis has provided hitherto unavailable information on contact lens deposition and its influence on subjective symptoms and bacterial binding. These results suggest that protein deposition has a significant potential to cause problems. Therefore, it is important that practitioners advise their patients regarding the importance of lens disinfection and cleaning and appropriate lens replacement schedules. These results will also be useful for the contact lens industry and the general field of biomaterials research.

Over 60 proteins have been detected in the tear film and among these lysozyme has attracted the greatest attention. Several techniques for elucidating the identity, quantity and conformation of lysozyme deposited on soft contact lenses have been developed. Lysozyme also deposits on the newly introduced silicone hydrogel (SH) lens materials, but in extremely low levels compared to conventional hydrogel lenses. Hence, a major analytical complication with the study of the SH contact lens materials relates to the minute quantity of deposited lysozyme. The first project of this thesis involved the development of a method whereby lysozyme mass extracted from SH lens materials would be preserved over time and would be compatible with an optimized Western blotting procedure. This methodological development was incorporated into a clinical study (CLENS-100® and Silicone Hydrogels ? CLASH study) wherein the difference in the degree of total protein, the difference in lysozyme deposition and activity recovered from lotrafilcon A SH lens material when subjects used surfactant containing rewetting drops (CLENS-100®) versus control saline was investigated. The remaining experiments were in vitro experiments wherein the lenses were doped in artificial lysozyme solution containing ¹²⁵I-labeled lysozyme. These experiments were performed to gain insight into the kinetics of lysozyme deposition on SH lens materials and also the efficacy of a reagent in extracting lysozyme from SH lens materials. A protocol was developed whereby the percentage loss of lysozyme mass found on lotrafilcon A SH lenses was reduced from approximately 33% to <1% (p<0. 001), following extraction and resuspension. The results from the CLASH study demonstrated that when subjects used a surfactant containing rewetting drop instead of a control saline drop total protein deposition (1. 2±0. 7 µg/lens versus 1. 9±0. 8 µg/lens, p<0. 001), lysozyme deposition (0. 7±0. 5 µg/lens versus 1. 1±0. 7 µg/lens, p<0. 001) and percentage lysozyme denaturation (76±10% versus 85±7%, p=0. 002) were all reduced. The results from the kinetics study demonstrated that lysozyme accumulated rapidly on etafilcon A lenses (1 hr, 98±8 µg/lens), reached a maximum on the 7th day (1386±21 µg/lens) and then reached a plateau (p=NS). Lysozyme accumulation on FDA Group II and SH lenses continued to increase across all time periods, with no plateau being observed (p<0. 001). The results from the extraction efficiency study showed that 0. 2% trifluoroacetic acid/ acetonitrile was 98. 3±1. 1% and 91. 4±1. 4% efficient in extracting lysozyme deposited on etafilcon A and galyfilcon lenses, while the lysozyme extraction efficiency was 66. 3±5. 3 % and 56. 7±3. 8% for lotrafilcon A and balafilcon lens materials (p<0. 001). The results from these studies re-emphasize that novel SH lens materials are highly resistant to protein deposition and demonstrate high levels of biocompatibility.

Introduction Contact lenses suffer from the same problems of deposition that other biomaterials exhibit, being rapidly coated with a variety of proteins, lipids and mucins. The first event observed at the interface between a contact lens and tear fluid is protein adsorption. Protein deposits on contact lenses are associated with diminished visual acuity, dryness and discomfort and lid-related inflammatory changes. The aim of this thesis was to determine the quantity and the conformational state of lysozyme deposited on contact lens materials over various time periods and also to determine the clinical relevance of protein deposits on contact lenses. The specific aims of each chapter of this thesis were as follows: • Chapter 4: To determine the total lysozyme deposition on conventional and silicone hydrogel contact lens materials as a function of time by artificially doping lenses with 125I-labeled lysozyme. • Chapter 5: To determine the conformational state of lysozyme deposited on conventional and silicone hydrogel contact lens materials as a function of time using an in vitro model. • Chapter 6: To quantify the total protein, total lysozyme and the conformational state of lysozyme deposited on a novel, lathe-cut silicone hydrogel contact lens material after three-months of wear. • Chapter 7: To determine the relationship between protein deposition and clinical signs & symptoms after one-day wear of etafilcon lenses in a group of symptomatic and asymptomatic lens wearers. • Chapter 8: To determine the influence of individual tear proteins (lysozyme, lactoferrin and albumin) on the adhesion of Gram positive and Gram negative bacteria to conventional and silicone hydrogel contact lens materials. Methods • Chapter 4: Conventional hydrogel FDA group I (polymacon), group II (alphafilcon A and omafilcon A), group IV (etafilcon A and vifilcon A), polymethyl methacrylate and silicone hydrogel lens materials (lotrafilcon A, lotrafilcon B, balafilcon A, galyfilcon A and senofilcon A) were incubated in a lysozyme solution containing 125I-labeled lysozyme for time periods ranging from 1 hour to 28 days. After each time period, lysozyme deposited on contact lens materials was determined using a Gamma Counter. • Chapter 5: Conventional hydrogel FDA groups I, II, IV and silicone hydrogel lens materials were incubated in lysozyme solution for time periods ranging from 1 hour to 28 days. After each time period, the lysozyme deposited on the lenses was extracted and the sample extracts were assessed for lysozyme activity and total lysozyme. • Chapter 6: 24 subjects completed a prospective, bilateral, daily-wear, nine month clinical evaluation in which the subjects were fitted with a novel, custom-made, lathe-cut silicone hydrogel lens material (sifilcon A). After 3 months of wear, the lenses were collected and total protein, total lysozyme and active lysozyme deposition were assessed. • Chapter 7: 30 adapted soft contact lens wearers (16 symptomatic and 14 asymptomatic) were fitted with etafilcon lenses. Objective measures and subjective symptoms were assessed at baseline and after hours 2, 4, 6 and 8. After 2, 4, 6 and 8 hour time points, lenses were collected and total protein, total lysozyme and active lysozyme deposition were assessed. • Chapter 8: Three silicone hydrogel (balafilcon A, lotrafilcon B & senofilcon A) and one conventional hydrogel (etafilcon A) lens materials were coated with lysozyme, lactoferrin and albumin. Uncoated and protein-coated contact lens samples were incubated in a bacterial suspension of Staphylococcus aureus 31 and two strains of Pseudomonas aeruginosa (6294 & 6206). The total counts and the viable counts of the adhered bacteria were assayed. Results • Chapter 4: Lysozyme accumulated rapidly on conventional hydrogel FDA group IV lenses, reached a maximum on day 7 and then plateaued with no further increase. PMMA showed a deposition pattern similar to that seen on lotrafilcon A and lotrafilcon B silicone hydrogel lenses. After 28 days, conventional hydrogel FDA group IV lenses deposited the most lysozyme. • Chapter 5: After 28 days, lysozyme deposited on group IV lenses exhibited the greatest activity. Lysozyme deposited on polymacon, lotrafilcon A and lotrafilcon B exhibited the lowest activity. Lysozyme deposited on omafilcon, galyfilcon, senofilcon, and balafilcon exhibited intermediate activity. • Chapter 6: The total protein recovered from the custom-made lenses was 5.3±2.3 µg/lens and the total lysozyme was 2.4±1.2 µg/lens. The denatured lysozyme found on the lenses was 1.9±1.0 µg/lens and the percentage of lysozyme denatured was 80±10%. • Chapter 7: Correlations between subjective symptoms and protein deposition showed poor correlations for total protein/ lysozyme and any subjective factor, and only weak correlations between dryness and active lysozyme. However, stronger correlations were found between active lysozyme and subjective comfort. • Chapter 8: Different tear proteins had varying effects on the adhesion of bacteria to contact lens materials. Lysozyme deposits on contact lenses increased the adhesion of Gram positive Staphyloccocus aureus 31 strain, while albumin deposits increased the adhesion of both the Gram positive Staphyloccocus aureus and Gram negative Pseudomonas aeruginosa 6206 & 6294 strains. Lactoferrin deposits increased the total counts of both the Gram positive and Gram negative strains, while they reduce the viable counts of the Gram negative strains. Conclusions • Chapter 4: Lysozyme deposition is driven by both the bulk chemistry and also the surface properties of conventional and silicone hydrogel contact lens materials. The surface modification processes or surface-active monomers on silicone hydrogel lens materials also play a significant role in lysozyme deposition. • Chapter 5: The reduction in the activity of lysozyme deposited on contact lens materials is time dependent and the rate of reduction varies between lens materials. This variation in activity recovered from lenses could be due to the differences in surface/ bulk material properties or the location of lysozyme on these lenses. • Chapter 6: Even after three-months of wear, the quantity of protein and the conformational state of lysozyme deposited on these novel lens materials was very similar to that found on similar surface-coated silicone hydrogel lenses after two to four weeks of wear. These results indicate that extended use of the sifilcon A material is not deleterious in terms of the quantity and quality of protein deposited on the lens. • Chapter 7: In addition to investigating the total protein deposited on contact lenses, it is of significant clinical relevance to determine the conformational state of the deposited protein. • Chapter 8: Uncoated silicone hydrogel lens materials bind more Gram positive and Gram negative bacteria than uncoated conventional hydrogel lens materials. Lysozyme deposited on contact lens materials does not possess antibacterial activity against all bacterial strains tested, while lactoferrin possess an antibacterial effect against certain Gram negative strains tested in this study. This thesis has provided hitherto unavailable information on contact lens deposition and its influence on subjective symptoms and bacterial binding. These results suggest that protein deposition has a significant potential to cause problems. Therefore, it is important that practitioners advise their patients regarding the importance of lens disinfection and cleaning and appropriate lens replacement schedules. These results will also be useful for the contact lens industry and the general field of biomaterials research.

This design covers the design of an integrated circuit (IC) in support of the active contact lens project at Cal Poly. The project aims to monitor intraocular eye pressure (IOP) to help diagnose and treat glaucoma, which is expected affect 6.3 million Americans by 2050. The IC is designed using IBM’s 130 nm 8RF process, is powered by an on-lens thin film 3.8 V rechargeable battery, and will be fabricated at no cost through MOSIS. The IC features a low-power linear regulator that powers a current-starved voltage-controlled oscillator (CSVCO) used for establishing a backscatter communication link. Additional circuitry is included to regulate power to and from the battery. An undervoltage lockout circuit protects the battery from deep discharge damage. When recharging, a rectifier and a voltage regulator provides overvoltage protection. These circuit blocks are biased primarily using a 696 mV subthreshold voltage reference that consumes 110.5 nA.

No description available.