A Novel Use of Confocal Microscopy to Study Lysozyme Sorption to Silicone Hydrogel and Conventional Hydrogel Contact Lens Materials

Abstract

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.