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Guillaume Normand, Jan Penraat, Joy Ghosh, Kellyann Kovach, Mikele Simkins, Michael Rosol, Cynthia L Grosskreutz, Sudeep Chandra; A novel imaging technique to non-invasively track molecules in the eye. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4115.
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© ARVO (1962-2015); The Authors (2016-present)
Understanding biodistribution in the eye is a critical step in developing novel therapeutics, yet outside of small animals the ability to visualize such distribution remains limited. A novel in vivo imaging method was evaluated to non-invasively and longitudinally measure the distribution of molecules after intravitreal (IVT) injection. The goals of this study were to select the optimal dye for in vivo use, to build a quantification method and to develop a translatable technology.
The near-infrared probes IRDye800CW and Indocyanine Green (ICG), as well as fluorescein, were evaluated ex vivo and in vivo after IVT or intravenous (IV) dosing. Cynomolgus monkeys were taken to the imaging room under anesthesia. After pupil dilation, animals were placed in the prone position and images were captured with a Spectralis, a confocal laser ophthalmoscope (cSLO), using 30 degree and ultra-widefield lenses (UWF) and at different sensitivities and diopters. Fluorescence intensity was measured at the retina and in the vitreous using the CellProfiler software.
IRDye800CW was found to be brighter both ex vivo and in vivo and less prone to quenching at high concentrations as compared to ICG. Moreover, IRDye800CW was shown to be cleared less rapidly than fluorescein and ICG from the retina and blood pool after IVT or IV injection, respectively. Based on a dose comparison study, the minimum detectable concentration of IRDye800CW at the retina was determined to be between 0.0349ug and 0.349ug. After IVT injection of 3.49ug of IRDye800CW, fluorescence intensity at the retina peaked 24h post-dose but was undetectable after 15 days post-dose. Interestingly, IRDye8000CW was observed to accumulate in the optic disc as well as form speckles along the retinal arteries. The UWF lens was able to demonstrate the high variability of the injection site and pattern by capturing a larger area of the retina.
Of the dyes tested, IRDye800CW was validated in this study as the optimal dye for in vivo ocular imaging and potential future clinical use. Distribution of the free dye was measured in several animals in order to benchmark the passage of the free dye as compared to other targeted probes. In contrast to other methods, this is a non-invasive technique that combines qualitative as well as semi-quantitative measurements of a molecule’s vitreous passage and provides useful anatomical information, especially for the retina.
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