Protein Deposition and Bacterial Adhesion to Conventional and Silicone Hydrogel Contact Lens Materials

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

Lysozyme Deposition Studies on Silicone Hydrogel Contact Lens Materials

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

Impression Cytology of the Lid Wiper Area

Muntz, A., van Doorn, K., Subbaraman, L. N., & Jones, L. W. (2016). Impression Cytology of the Lid Wiper Area. Journal of Visualized Experiments, (114). https://doi.org/10.3791/54261Few reports on the cellular anatomy of the lid wiper (LW) area of the inner eyelid exist and only one report makes use of cytological methods. The optimization of a method of collecting, staining and imaging cells from the LW region using impression cytology (IC) is described in this study. Cells are collected from the inner surface of the upper eyelid of human subjects using hydrophilic polytetrafluoroethylene (PTFE) membranes, and stained with cytological dyes to reveal the presence of goblet cells, mucins, cell nuclei and various degrees of pre- and para-keratinization. Immunocytochemical dyes show cell esterase activity and compromised cell membranes by the use of a confocal scanning laser microscope. Up to 100 microscopic digital images are captured for each sample and stitched into a high-resolution, large scale image of the entire IC span. We demonstrate a higher sensitivity of IC than reported before, appropriate for identifying cellular morphologies and metabolic activity in the LW area. To our knowledge, this is the first time this selection of fluorescent dyes was used to image LW IC membranes. This protocol will be effective in future studies to reveal undocumented details of the LW area, such as assessing cellular particularities of contact lens wearers or patients with dry eye or lid wiper epitheliopathy

Release of Ciprofloxacin and Moxifloxacin From Daily Disposable Contact Lenses From an In Vitro Eye Model

Bajgrowicz, M., Phan, C.-M., Subbaraman, L. N., & Jones, L. (2015). Release of Ciprofloxacin and Moxifloxacin From Daily Disposable Contact Lenses From an In Vitro Eye Model. Investigative Opthalmology & Visual Science, 56(4), 2234. https://doi.org/10.1167/iovs.15-16379Purpose.: To analyze the release of two fluoroquinolones, ciprofloxacin and moxifloxacin, from conventional hydrogel (CH) and silicone hydrogel (SH) daily disposable contact lenses (CLs), comparing release from a fixed-volume vial and a novel in vitro eye model. Methods.: Four CH CLs (nelfilcon A, omafilcon A, etafilcon A, ocufilcon B) and three SH CLs (somofilcon A, narafilcon A, delefilcon A) were used. The lenses were incubated in drug solutions for 24 hours. After the incubation period, the lenses were placed in two release conditions: (1) a vial containing 4.8 mL PBS for 24 hours and (2) an in vitro eye model with a flow rate at 4.8 mL over 24 hours. Results.: Release in the vial for both drugs was rapid, reaching a plateau between 15 minutes and 2 hours for all lenses. In contrast, under physiological flow conditions, a constant and slow release was observed over 24 hours. The amounts of ciprofloxacin released from the lenses ranged between 49.6 ± 0.7 and 62.8 ± 0.3 μg per lens in the vial, and between 35.0 ± 7.0 and 109.0 ± 5.0 μg per lens in the eye model. Moxifloxacin release ranged from 24.0 ± 4.0 to 226.0 ± 2.0 μg per lens for the vial, and between 13.0 ± 2.0 and 151.0 ± 10.0 μg per lens in the eye model. In both systems and for both drugs, HEMA-based CLs released more drugs than other materials. Conclusions.: The parameters of the release system, in particular the volume and flow rate, have a significant influence on measured release profiles. Under physiological flow, release profiles are significantly slower and constant when compared with release in a vial

Release of Moxifloxacin from Contact Lenses Using an In Vitro Eye Model: Impact of Artificial Tear Fluid Composition and Mechanical Rubbing

Phan, C.-M., Bajgrowicz-Cieslak, M., Subbaraman, L. N., & Jones, L. (2016). Release of Moxifloxacin from Contact Lenses Using an In Vitro Eye Model: Impact of Artificial Tear Fluid Composition and Mechanical Rubbing. Translational Vision Science & Technology, 5(6), 3. https://doi.org/10.1167/tvst.5.6.3Purpose: The aim of this study was to evaluate and compare the release of moxifloxacin from a variety of daily disposable (DD) contact lenses (CLs) under various conditions using a novel in vitro eye model. Methods: Four commercially available DD conventional hydrogel (CH) CLs (nelfilcon A, omafilcon A, etafilcon A, and ocufilcon B) and three silicone hydrogel (SH) CLs (somofilcon A, narafilcon A, and delefilcon A) were evaluated. These lenses were incubated in moxifloxacin for 24 hours. The release of the drug was measured using a novel in vitro model in three experimental conditions: (1) phosphate buffered saline (PBS); (2) artificial tear solution (ATS) containing a variety of proteins and lipids; and (3) ATS with mechanical rubbing produced by the device. Results: Overall, CH CLs had a higher drug release than SH CLs (P < 0.05) under all conditions. Typically, a higher drug release was observed in PBS than ATS (P < 0.05). For CH, drug release was found to be higher in ATS with rubbing than PBS or ATS (P < 0.05). For most lens types, ATS with rubbing produced higher drug release than ATS alone (P < 0.05). Generally, the release kinetics for all conditions were sustained over the 24-hour testing period, and no burst release was observed (P < 0.05). Conclusions: Moxifloxacin release from a CL into ATS is lower when compared to release into PBS. When mechanical rubbing is introduced, the amount of drugs released is increased. Translational Relevance: Results suggest that sophisticated in vitro models are necessary to adequately model on-eye drug release from CL materials

Insights to Using Contact Lenses for Drug Delivery

Phan, C.-M. (2013). Insights to Using Contact Lenses for Drug Delivery. Clinical & Experimental Pharmacology, 04(01). https://doi.org/10.4172/2161-1459.1000145There has been considerable interest in the potential application of contact lenses for ocular drug delivery. This short communication provides an overview of the challenges faced by delivering drugs using contact lenses, highlights the solutions to limitations that have already been achieved, and describes the barriers that remain before commercial application can be realized.NSERC 20/20 Network for the Development of Advanced Ophthalmic Material

Development of an In Vitro Ocular Platform to Test Contact Lenses

The definitive version of Development of an in Vitro Ocular Platform to Test Contact Lenses was published by Emerald www.emeraldinsight.com in Journal of Visualized Experiments, (110) (2016) https://doi.org/10.3791/53907Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic physiological tear volumes.7 The traditional model also lacks the natural tear flow component and the blinking reflex, both of which are defining factors of the ocular environment. The development of a novel model is described in this study, which consists of a unique 2-piece design, eyeball and eyelid piece, capable of mimicking physiological tear volume. The models are created from 3-D printed molds (Polytetrafluoroethylene or Teflon molds), which can be used to generate eye models from various polymers, such as polydimethylsiloxane (PDMS) and agar. Further modifications to the eye pieces, such as the integration of an explanted human or animal cornea or human corneal construct, will permit for more complex in vitro ocular studies. A commercial microfluidic syringe pump is integrated with the platform to emulate physiological tear secretion. Air exposure and mechanical wear are achieved using two mechanical actuators, of which one moves the eyelid piece laterally, and the other moves the eyeballeyepiece circularly. The model has been used to evaluate CLs for drug delivery and deposition of tear components on CLs

The TFOS international workshop on contact lens discomfort: report of the contact lens materials, design, and care subcommittee

Jones, L., Brennan, N. A., González-Méijome, J., Lally, J., Maldonado-Codina, C., Schmidt, T. A., … Nichols, J. J. (2013). The TFOS International Workshop on Contact Lens Discomfort: Report of the Contact Lens Materials, Design, and Care Subcommittee. Investigative Opthalmology & Visual Science, 54(11), TFOS37. https://doi.org/10.1167/iovs.13-13215Examining the role of the contact lens material, design, and the care system is fundamental to understanding contact lens discomfort (CLD). However, a systematic review that tries to determine the governing factors is fraught with difficulties. A lack of a validated “instrument” (or single validated questionnaire) for measuring discomfort makes it impossible to compare between studies because reported levels of comfort (or discomfort) are inconsistent. Subject classifications can vary widely, from studies that include only neophytes or asymptomatic contact lens (CL) wearers to studies including only those contact lens–wearing subjects who experience marked dryness or symptoms of discomfort. Also, it is difficult to measure issues of importance in isolation because changing one factor in a contact lens or care solution can invariably affect another. An illustration of this relates to a change in hydrogel water content, which also affects oxygen permeability, oxygen transmissibility, modulus, and possibly lens thickness. Finally, various confounding factors between studies also make true comparisons problematic. Typical examples would include differences between brands of lenses made from the same material (which may have differing geometric designs, edge configuration, or production methods); wearing modality (lenses may be worn on a daily wear [DW] basis, overnight occasionally, or for up to 30 nights on a continuous wear [CW] basis); duration of use prior to replacement, wearing time during the day (from just a few hours to most of the day); and care product differences or exposures (which could range from no exposure in the case of daily disposable [DD] materials to a preserved system that has extensive uptake and release from the contact lens material being examined). The purpose of this report is to summarize evidence-linking associations, mechanistic and etiological factors between contact lens materials, designs, and care solutions with CLD. The potential factors associated with this are many and varied, and graphically display the complexity of this issue

BCLA CLEAR Presbyopia: Management with contact lenses and spectacles

This paper seeks to outline the history, market situation, clinical management and product performance related to the correction of presbyopia with both contact lenses and spectacles. The history of the development of various optical forms of presbyopic correction are reviewed, and an overview is presented of the current market status of contact lenses and spectacles. Clinical considerations in the fitting and aftercare of presbyopic contact lens and spectacle lens wearers are presented, with general recommendations for best practice. Current options for contact lens correction of presbyopia include soft simultaneous, rigid translating and rigid simultaneous designs, in addition to monovision. Spectacle options include single vision lenses, bifocal lenses and a range of progressive addition lenses. The comparative performance of both contact lens and spectacle lens options is presented. With a significant proportion of the global population now being presbyopic, this overview is particularly timely and is designed to act as a guide for researchers, industry and eyecare practitioners alike

Lysozyme Deposition Studies on Silicone Hydrogel Contact Lens Materials

I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. ii 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 wa