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Steven Cheung, Lakshman N Subbaraman, Tannin A Schmidt, Lyndon William Jones; Localization of full-length recombinant human proteoglycan 4 in commercial contact lenses using confocal microscopy. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6059.
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Proteoglycan 4 (PRG4) is a boundary lubricant that can improve the wettability and lubricity of model contact lenses. The aim of this study was to determine the location of full-length recombinant human PRG4 (rh-PRG4) tagged with fluorescein isothiocyanate (FITC) in various commercial contact lenses, using a confocal laser scanning microscopy (CLSM) technique.
Four commercially available silicone hydrogel contact lenses [balafilcon A (BA, PureVision, Bausch+ Lomb), senofilcon A (SA, Acuvue Oasys, Vistakon), comfilcon A (CA, Biofinity, CooperVision), lotrafilcon B (LB, Air Optix, Alcon)] and one conventional hydrogel lens material [etafilcon A (EA, Acuvue 2, Vistakon)] were examined. Purified rh-PRG4, expressed in a proprietary Chinese hamster ovary cell line, was provided by Lubris, LLC and was manually tagged with FITC via amine reaction to obtain a labeling ratio of approximately 5-6 dye/protein. Unconjugated FITC was removed using a Sephadex-G25 resin in phosphate buffered saline (PBS). Lenses were incubated under two conditions: (1) rh-PRG4-FITC solution at 300 µg/ml and (2) PBS for 1 hour at 37°C in darkness with gentle shaking. After incubation, the central 4 mm of each lens was removed and fixed onto a microscope slide and viewed with the Zeiss 510 CLSM using the argon laser at 488 nm. Scans were taken at 1 µm intervals to a maximum depth of approximately 100 µm. Images were processed using the Zen 2009 software.
All lens materials demonstrated sorption of the rh-PRG4. SA revealed rh-PRG4-FITC penetrating into the bulk of the lens material uniformly, yet slightly favoring the two surfaces of the material. The same was observed with BA lenses, with a greater degree of rh-PRG4-FITC being seen at the surface compared to SA. Conversely, the rh-PRG4 was seen exclusively on the two surfaces of LB and having no presence within the bulk of the lens. For CA and EA lenses, rh-PRG4-FITC was evenly distributed throughout the bulk of the lens, as well as on the two surfaces.
The location of PRG4 conjugated with FITC in a contact lens can be successfully visualized using CLSM. The lens chemistry, such as polymer composition, surface treatment and hydrophobicity, can influence the sorption profile of rh-PRG4. This sorption profile may potentially impact rh-PRG4’s lubricating properties of lenses.
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