Lipids are essential tear components that aid the stability of the tear film (TF) to protect it from excess evaporation. The composition, conformation, and function of TF lipids are jeopardized by external factors such as contact lens (CL) wear and environmental elements (i.e. UV radiation, oxidation). Specifically, silicone hydrogel (SiHy) CLs exhibit relatively high deposition of TF lipids that may be associated with visual disturbances and discomfort. Additionally, lipids are degraded by oxidation and may cause alterations of the TF lipid layer, which in turn might be a source for dry eye symptoms.
The overall goal of this thesis was to evaluate the quantity and location of lipid deposition on various CL materials over time and also to assess the impact lipid contamination may have on various care products and TF quality measurements.
The specific aims of each chapter of this thesis were as follows:
• Chapter 3: To determine the efficacy of multi-purpose solutions (MPS) on the removal of cholesterol deposits from SiHy lens materials.
• Chapter 4: To analyze the uptake of cholesterol on SiHy and conventional hydrogel (CH) daily disposable (DD) CL materials using an in vitro radiochemical detection method.
• Chapter 5: To evaluate the differences in lipid uptake and penetration in DD CL using the conventional “in-vial” method compared to a novel in vitro eye model.
• Chapter 6: To develop a novel in vitro model to determine pre-lens non-invasive break- up times (NIBUT) and to subsequently compare the break-up times over five contemporary DD lens materials.
• Chapter 7: To optimize and develop a method to determine and quantify lipid peroxidation by-products that indicates oxidative stress in tears.
Materials and Methods
• Chapter 3: Five contemporary SiHy lens materials were incubated for 7 days using a radiochemical experiment. Additionally, lenses were stored and cleaned in different MPSs using a rub and rinse technique. Lipids were then extracted from lenses with 2:1 chloroform:methanol, analyzed in a beta-particle radiation counter and μg/lens of cholesterol was determined.
• Chapter 4: Seven different commercially available DD CLs were incubated for 16 hours to determine the impact of material composition on cholesterol deposition. Subsequent to the incubation, lenses were extracted using 2:1 chloroform:methanol and the extracts were analyzed in a beta-particle radiation counter and (ng/lens) extrapolated from standard curves.
• Chapter 5: Seven DD CLs were incubated for 4 and 12 hours in an artificial tear solution (ATS) containing fluorescently-labelled cholesterol (7-nitrobenz-2-oxa-1,3-diazol-4- yl-cholesterol, or NBD-cholesterol). Additionally, CLs were incubated in an “in-vial”
condition and compared to a novel in vitro eye platform, designed to simulate physiological tear flow, tear volume, and ‘simulated’ blinking. After the incubation period, the CLs were analyzed using a laser scanning confocal microscope (LSCM), and quantitative analyses for penetration depth and relative fluorescence intensity values were carried out.
• Chapter 6: Five DD lens materials were incubated in an artificial tear solution using a model blink cell that mimics intermittent air exposure. CLs were incubated by repeatedly being submerged and exposed to air for up to 16 hours. A corneal topographer (Topcon CA-100) was used to illuminate the lens surfaces with placido rings, which were captured with a digital video camera and from which NIBUTs of the CL materials were determined.
• Chapter 7: Tear samples were collected using calibrated disposable capillary tubes and various assays that quantify lipid peroxidation by-products were compared against each other: thiobarbituric acid reactive substances (TBARS) assay, malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and oxidized low-density-lipoproteins (OxLDL) enzyme-linked immunosorbent assays (ELISAs). Pooled and individual tear samples were diluted in a wide range to determine the lowest volume of tears that could be used. Subsequently, the fluorescence was measured with a fluorescence spectrophotometer at 530 nm (excitation) and 590 nm (emission), as well as their absorbance at 450 nm.
• Chapter 3: For all lens materials, only one of the multipurpose solutions removed more cholesterol than any other test solution; however, the amount of cholesterol removed from the individual CLs was statistically significant only for the two lens materials that deposited the most: balafilcon A (0.93±0.02μg/lens) and senofilcon A (0.95±0.01μg/lens). All of the other solutions evaluated showed no significant effect on lipid removal.
• Chapter 4: Cholesterol deposited significantly more on SiHy lenses than CHs. The uptake of cholesterol ranged from 22.63 ± 2.98 ng/lens to 97.94 ± 4.18 ng/lens for all lens materials, with narafilcon A accumulating the largest quantity of cholesterol. The accumulation of cholesterol was shown to be continuous throughout the 16 hours of incubation without reaching a plateau.
• Chapter 5: The depth of penetration of NBD-cholesterol varied between the vial and the eye-blink platform. In general, SiHy lenses showed higher intensities of NBD- cholesterol than CH materials and the fluorescence intensities also varied between the incubation methods as well as the lens materials.
• Chapter 6: Overall, NIBUTs ranged from 26.19 ± 5.79 s to 1.23 ± 0.13 s. After the initial (T0) break-up times were determined, CH CLs revealed significantly longer NIBUTs than SiHy CLs. After 16 hours of incubation, the SiHy lens material delefilcon A had the longest break-up time. Significant changes of NIBUTs within the lens materials varied between the examined time points. After 16 hours, all CLs showed significant reductions in NIBUTs in comparison to T0.
• Chapter 7: After tear samples were pooled and concentrated, 0.056±0.004 μM of MDA could be measured using the TBARS assay. After optimizing various ELISAs, OxLDL in individual tear samples (2.5μL) ranged between 45.59 ± 2.95 ng/mL and 28.24 ± 4.66 ng/mL. All measurements using the MDA- and 4-HNE ELISA were below the assays limit of detection.
• Chapter 3: Lipid-removal efficacy varies depending on the combination of lens material and solution. Only one MPS showed a significant reduction of lipids for any of the tested lens materials.
• Chapter 4: For the periods of time that DD lens materials are worn, cholesterol deposits significantly more onto SiHy lenses than CHs. This could have implications for wearers who have higher levels of lipid in their tears that are fitted with SiHy DD materials.
• Chapter 5: This study provides a novel in vitro approach to evaluate deposition and penetration of lipids on CLs. We show that the traditional “in-vial” incubation method exposes the CLs to an excessively high amount of ATS, which results in an overestimation for cholesterol deposition. The novel eye-platform, provides a more natural environment for in vitro lens incubation studies which will consequently better elucidate the interactions between CLs and TF components.
• Chapter 6: NIBUT values reduced gradually over time and varying levels of deposition on different CLs may impact the measured pre-lens NIBUT of various lens materials. While NIBUT of CH materials are longer than that obtained with SiHy materials immediately out of the blister pack, it appears that after TF exposure, the NIBUTs determined between CH and SiHy DD materials are very similar.
• Chapter 7: Assays for oxidative stress were optimized and showed that oxidative stress is detectible in small quantities of tears (2.5 μL). These techniques could be employed to determine oxidative stress in TF lipids, which could potentially help to identify patients with dry eye and CL discomfort.
This thesis has provided previously unavailable information on lipid deposition on CLs and its effect on clinically relevant TF quality measurements. The results showed that current CL cleaning solutions fail to efficiently remove lipid contaminations and that DD SiHy lens materials provide options for clinicians to consider when patients experience complications and excess lipid uptake with daily wear lens materials. Furthermore, this thesis has presented novel in vitro methods that will be useful for other researchers and the CL industry to appropriately test and predict CL performance.||en