Tumor hypoxia fluorescence imaging using 2-nitroimidazole bis-carboxylic acid indocyanine dye conjugate

We present tumor hypoxia mapping by diffuse optical fluorescence tomography. A novel 2-nitroimidazole bis-carboxylic acid indocyanine dye conjugate has been developed for tumor-targeted hypoxia fluorescence imaging. The hypoxia probe has been evaluated in-vitro using 4T1 tumor cell lines and in-vivo tumor targeting in mice. In-vivo tumor targeting in six mice demonstrated that a measured half-life of 2-nitroimidazole-indocyanine dye wash out in the tumor was significantly longer (112±32.37 minutes) than that of bis-carboxylic acid indocyanine dye (69.75±14.01 minutes). The bis-carboxylic acid indocyanine dye was completely washed out from the tumor site within 3-5 hours post-injection, but 2-nitroimidazole-ICG remained for up to 21 hours in the tumor site. Near infrared fluorescence images of mice tumors showed a 2.6-fold contrast of dye uptake with hypoxic conjugate injection (7.46±1.68 μM) compared to that with indocyanine dye injection (2.9±0.60 μM). The in-vitro cell studies were performed to assess fluorescence labeling comparing hypoxia to normoxia conditions. A fluorescence emission ratio of 2.5-fold was found between the cells treated with the 2-nitroimidazole-indocyanine dye and incubated under hypoxia compared to the cells in normoxia condition. Hypoxia specificity was also confirmed when compared to cells treated with unconjugated indocyanine dye alone. Fluorescence images acquired using a Li-COR scanner from harvested tumor samples support the in vivo monitoring and imaging results.