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S107: Phototoxicity Testing of Emerging Fluorescence Imaging Products: Reactive Oxygen Species Generation and Photocytotoxicity

Poster Presenter

Shruti Vig

Graduate Student
University of Maryland, College Park
United States

Objectives

The objective of this abstract is to assess the phototoxicity of fluorophores used in optical imaging procedures using commercial cell-free assay and modified 3T3 Neutral Red Uptake (3T3 NRU) assessment, with the aim of standardizing photosafety testing for clinical fluorescence imaging products.

Method

A cell-free chemical assay measuring singlet oxygen (SO) generation and a modified 3T3 NRU assay were used to evaluate the dose-response curve for each test agent. Measurements were taken at concentrations and illumination parameters within and above clinical levels.

Results

Four imaging agents – ICG, Proflavine, Methylene Blue (MB), and IRDye800 – and three strong reactive molecular species (RMS)-generating fluorophores – IRDye700, BPD, and Rose Bengal (RB)– were evaluated. For the chemical assay, SO production rate was calculated and compared data from the literature. For photocytotoxicity assessment, a 3T3 NRU assay was performed to evaluate the dose-response curve for each agent. Additionally, Mean Photo Effect (MPE) and the Photo Irritation Factor (PIF) values were calculated using the Phototox Version 2.0 software for each agent. Results indicate that SO production was highly dependent on the radiant exposure and agent concentration. SO production rates for ICG and IRDye800 were more than two orders of magnitude below that of RB. MB and IRDye700 showed high levels of SO production. Proflavine exhibited a spectral overlap with the cell-free chemical assay, yet our tests indicated minimal SO production and appeared robust due to the use of a background subtraction approach. Our results with the modified 3T3 NRU assay exhibited expected predictions of photocytotoxicity, including for known phototoxic agents, BPD and RB. However, Proflavine exhibited photocytotoxicity using the 3T3 NRU assay despite the lack of SO generation with the cell-free assay. This may be due to the production of other RMS – such as superoxide and hydroxyl radicals which could not be detected with the SO-specific cell-free chemical assay. Overall, our results for the two methods appeared consistent and in agreement with the literature.

Conclusion

Using these methods to estimate RMS production and related photocytotoxicity should provide practical insights into the photochemical safety of contrast-enhanced fluorescence imaging products. The methods implemented deviate from standard approaches in that they focus on generating results for clinically relevant light and agent doses. While the study focused on quantifying SO production, additional assays will be needed to detect other known RMS produced by fluorophores to establish a comprehensive test methodology. Additionally, evaluating overall photocytotoxicity produced due to collective RMS generation using the in-vitro 3T3 NRU assay may help determine a safe concentration and illumination dose range for clinical imaging. Results from this study provide critical scientific information and help establish consensus standards and regulatory guidelines for innovative diagnostic technologies that are safe for clinical use and hence benefit public health.