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Shengyan Liu, Aya Tsugimatsu, Chu-Ning Chang, Huiyuan Liang, Matthew-Mina Reyad, Shelley Boyd, Lyndon William Jones, Frank Gu; Tracking ocular retention of precorneal formulations using near-infrared dyes. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4444.
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There is much interest in developing new fluorescence tools for tracking precorneal activities. Fluorescein based dyes are often used to analyze precorneal retention of materials but they are unreliable due to photobleaching and the irritation caused by the method. We hypothesize that a near infrared (NIR) dye labeling method is ideal for tracking precorneal activities due to higher photostability and the invisibility of the NIR excitation beam.
We used a confocal scanning laser ophthalmoscope (cSLO) to detect dye-labelled materials on the precorneal surfaces of male NZW rabbits. We compared the retention between dextran and dextran modified with phenylboronic acid (PBA), a mucosal targeting ligand, by labelling them with either blue-light dyes such as fluorescein or Alexa Fluor 488 (AF), or with the NIR dye Cyanine 7.5. One eye of each rabbit was administered a control formulation (dye-labelled dextran), while the contralateral eye was administered a PBA-modified dextran (dextran-PBA) labelled with the same dye (n = 3). Each eye was manually blinked 3x before capturing initial images (0 hr). Subsequent images were obtained at 3, 6, 9 and 24 hrs.
NIR dye-labelled dextran-PBA showed improved retention at 3 hrs compared to the same dye-labelled dextran (p < 0.05) and continued to show visible ocular fluorescence for up to 24 hrs, demonstrating the mucoadhesive property of PBA ligands. However, both dextran and dextran-PBA labelled with blue-light dyes did not show any visible fluorescence on the rabbit eyes beyond 3 hrs, likely due to photobleaching. In addition, since fluorescein and AF require blue light for excitation, the animals were irritated by the high intensity blue light during imaging leading to behaviours such as flinching, squinting, blinking, which confounded the clearance of ocular formulations. In comparison, the NIR light goes largely undetected by the rabbits, eliminating this confound, maintaining normal blinking rates and providing more accurate ocular retention data for the formulation.
Photobleaching and irritation caused unreliable ocular retention results while using a blue fluorescent dye labeling method. We conclude that NIR dyes are ideal for ocular retention analysis of materials due to their higher physiological photostability and the invisibility of NIR excitation beam.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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