Abstract 2379: Tunable squaraine dyes as contrast agents for image guided surgery with optoacoustic imaging
Authors: MacCuaig William M. , Wickizer Carly , Henary Maged , Shao Yihan , Edil Barish H. , Jain Ajay , Grizzle William E. , McNally Lacey R. 
Published: April 2023

Surgical removal of cancers results in the most favorable patient outcomes. Resection margins may be tumor-positive in up to 70% of cases depending on cancer type, representing an unmet clinical need. Image-guided surgery often utilizes fluorescent dyes such as IR 800CW, but are limited to 8mm of depth and result in potential false-positive signal due to high blood binding. To overcome limitations, we are developing new contrast agents for Multispectral optoacoustic tomography (MSOT)-guided surgery to allow for greater depth of penetration and future potential of multiplexing of agents.To generate squaraine contrast agents, we prepared heterocyclic salts side arms refluxed with squaric acid to form 6 different compounds. The compounds differ only in halogenation of heterocyclic salts and inclusion of dual trimethylpropylammonium (TMAB) pendant groups. All squaraines were confirmed with NMR/spectrometry. MSOT and fluorescence were utilized to investigate the in vitro optoacoustic/fluorescence activity of the compounds. Computational modeling of squaraines through density functional theory was used to reveal quantum properties of the compounds including vibrational entropy, oscillator strength, and dipole moment. Compounds were administered orally in a murine model to confirm visualization capability with MSOT and fluorescence.Squaraine dyes functionalized with heavier halogens (Br, Cl) exhibited higher optoacoustic activity than dyes with less heavy (F), or without halogen. Specifically, TMAB/Br functionalized squaraine exhibited 2.12 optoacoustic units in vivo, compared to 0.81, 0.58, and 0.44 for Cl, F, and no halogen compounds, respectively (all p&lt;0.001). Inclusion of the dual TMAB groups increased optoacoustic activity. When comparing Br compounds with/without TMAB, the TMAB functionalized compound outperformed the counterpart significantly, (2.12 a.u. vs. 0.21 a.u., p&lt;0.001). Fluorescence intensity in vivo between TMAB/Br and TMAB/Cl compounds were not significantly different (3.07E9 vs. 2.81E9 counts), indicating that fluorescence signal does not necessarily predict optoacoustic activity. Computational modeling revealed heavy halogens and TMAB functionalized dyes exhibit increased vibrational entropy, oscillator strength, dipole moment, and presence of right-shifted absorbance peaks. In vivo studies in a murine model confirmed that heavy halogen and TMAB functionalized dyes were visible in the gastrointestinal tract using both MSOT and fluorescence imaging.Image-guided surgical removal of cancer yields best patient outcomes, but is currently limited by blood binding and imaging depth. MSOT is a potential candidate, but lack of contrast has hurt clinical application. This study focused on synthesis and evaluation of squaraine compounds as potential optoacoustic contrast to expand the potential of MSOT in a clinical setting for image guided surgery for cancer.

Citation Format: William M. MacCuaig, Carly Wickizer, Maged Henary, Yihan Shao, Barish H. Edil, Ajay Jain, William E. Grizzle, Lacey R. McNally. Tunable squaraine dyes as contrast agents for image guided surgery with optoacoustic imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2379.