Thomas, Chloe N; Alfahad, Nada; Capewell, Nicholas; Cowley, Jamie; Hickman, Eleanor; Fernandez, Antonio; Harrison, Neale; ... Hill, Lisa J; + view all Thomas, Chloe N; Alfahad, Nada; Capewell, Nicholas; Cowley, Jamie; Hickman, Eleanor; Fernandez, Antonio; Harrison, Neale; Qureshi, Omar S; Bennett, Naomi; Barnes, Nicholas M; Dick, Andrew D; Chu, Colin J; Liu, Xiaoxuan; Denniston, Alastair K; Vendrell, Marc; Hill, Lisa J; - view fewer (2022) Triazole-derivatized near-infrared cyanine dyes enable local functional fluorescent imaging of ocular inflammation. Biosensors and Bioelectronics , 216 , Article 114623. 10.1016/j.bios.2022.114623.

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Abstract

Near-infrared (NIR) chemical fluorophores are promising tools for in-vivo imaging in real time but often succumb to rapid photodegradation. Indocyanine green (ICG) is the only NIR dye with regulatory approval for ocular imaging in humans; however, ICG, when employed for applications such as labelling immune cells, has limited sensitivity and does not allow precise detection of specific inflammatory events, for example leukocyte recruitment during uveitic flare-ups. We investigated the potential use of photostable novel triazole NIR cyanine (TNC) dyes for detecting and characterising activated T-cell activity within the eye. Three TNC dyes were evaluated for ocular cytotoxicity in-vitro using a MTT assay and optimised concentrations for intraocular detection within ex-vivo porcine eyes after topical application or intracameral injections of the dyes. TNC labelled T-cell tracking experiments and mechanistic studies were also performed in-vitro. TNC-1 and TNC-2 dyes exhibited greater fluorescence intensity than ICG at 10 μM, whereas TNC-3 was only detectable at 100 μM within the porcine eye. TNC dyes did not demonstrate any ocular cell toxicity at working concentrations of 10 μM. CD4+T-cells labelled with TNC-1 or TNC-2 were detected within the porcine eye, with TNC-1 being brighter than TNC-2. Detection of TNC-1 and TNC-2 into CD4+T-cells was prevented by prior incubation with dynole 34-2 (50 μM), suggesting active uptake of these dyes via dynamin-dependent processes. The present study provides evidence that TNC dyes are suitable to detect activated CD4+T-cells within the eye with potential as a diagnostic marker for ocular inflammatory diseases.

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