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Despite the large interest in nuclear/optical multimodality imaging, the effect of radiation on the fluorescence of fluorophores remains largely unexplored. Herein, we report on the radiobleaching of cyanine fluorophores and describe conditions to provide robust radioprotection under practical (pre)clinical settings. We determined the radiosensitivity of several cyanine fluorescent compounds, including IRDye 800CW (800CW) and a dual modality imaging tetrapeptide containing DOTA as chelator and Dylight 800 as fluorophore, exposed to increasing activities of 111 In, 68 Ga, or 213 Bi (γ, EC/β, and α emitter, respectively). An activity and type of radiation-dependent radiation-induced loss of fluorescence, radiobleaching, of 800CW was observed upon incubation with escalating activities of 111 In, 68 Ga, or 213 Bi. 68 Ga showed the largest radiolytic effect, followed by 111 In and 213 Bi. The addition of oxygen radical scavengers including ethanol, gentisic acid, and ascorbic acid (AA), provided a concentration dependent radioprotective effect. These results supported the hypothesis of a free radical-mediated radiobleaching mechanism. AA provided the most robust radioprotection over a wide range of concentrations and preserved fluorescence at much higher radioactivity levels. Overall, both near-infrared fluorescent compounds displayed similar sensitivity, except for 213 Bi-irradiated solutions, where the dual modality construct exhibited enhanced radiolysis, presumably due to direct radiation damage from α particles. Concurrently, AA was not able to preserve fluorescence of the dual-modality molecule labeled with 213 Bi. Our findings have important consequences for several research areas including ROS sensing, radiation-mediated drug release (uncaging), fluorescent dosimetry, and in the preparation of dual-modality radiopharmaceuticals.

Preventing Radiobleaching of Cyanine Fluorophores Enhances Stability of Nuclear/NIRF Multimodality Imaging Agents