Impact of in vitro Tear Film Composition on Lysozyme Deposition and Denaturation
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Purpose To study the impact of lactoferrin and lipids on the kinetic deposition and denaturation of lysozyme on contact lens materials. Methods The contact lenses investigated in this thesis included 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]. All lenses were incubated in four solutions: a complex artificial tear solution (ATS); an ATS without lactoferrin; an ATS without lipids; and an ATS without lactoferrin and lipids. Following various time points, all lenses were prepared for lysozyme analysis using the methods below: • To quantify the kinetic uptake of lysozyme to different contact lens materials, I125-radiolabelled lysozyme was added to each incubation solution. Total lysozyme deposition was quantified using a gamma counter. • To study the activity of lysozyme deposited to contact lenses, a fluorescence-based lysozyme activity assay was compared to a turbidity assay. Potential interactions with lens materials and extraction solvents were evaluated. • To investigate the kinetic denaturation of lysozyme deposited to different contact lens materials, the fluorescence-based activity assay and the enzyme-linked immunosorbent assay were used. Results The presence of lactoferrin and lipids decreased lysozyme uptake to lotrafilcon B. Lysozyme deposition on senofilcon A was greater in the absence of lipids after day 21, however the opposite was seen with etafilcon A, where lysozyme uptake was lower without lipids in the ATS. Lactoferrin and/or lipids had no effect on lysozyme adsorption to omafilcon A. The fluorescence-based lysozyme activity assay demonstrated high sensitivity and a wide linear range of detection, which covers the amount of lysozyme typically extracted from contact lenses. Using this assay, lysozyme activity on both silicone hydrogel materials was lower in the presence of lipids in the ATS. In addition, lactoferrin had a protective effect on lysozyme activity for lysozyme sorbed to senofilcon A. Moreover, the presence of lactoferrin and/or lipids did not exhibit any effect on lysozyme denaturation with conventional hydrogel lenses. Conclusions The presence of lactoferrin and lipids in an artificial tear solution impacted lysozyme deposition and denaturation of lysozyme on various contact lenses. It is important for in vitro studies, when developing tear film models, to consider the effects of tear film components when investigating protein deposition and denaturation on contact lenses.
Cite this version of the work
Alan Ng (2012). Impact of in vitro Tear Film Composition on Lysozyme Deposition and Denaturation. UWSpace. http://hdl.handle.net/10012/6961