Engineering Phosphinate-Containing Rhodamines for Turn-On Photoacoustic Imaging Applications

Photoacoustic imaging (PAI) is an emerging imaging technique with applications in preclinical and point-of-care settings. PAI is a light-in, sound-out technique which uses pulsed laser excitation with near-infrared (NIR) light to elicit local temperature increases through non-radiative relaxation events, ultimately leading to the production of ultrasound waves. The classical xanthene dye scaffold has found numerous applications in fluorescence imaging, however, xanthenes are rarely utilized for PAI since they do not typically display NIR absorbance. Herein, we report the ability of Nebraska Red (NR) dyes to produce photoacoustic (PA) signal and provide a rational design approach to reduce the hydrolysis rate of ester containing dyes. By converting a relatively hydrolytically labile phosphinate ester to a more stable thiophosphinate ester, we were able to reduce the rate of ester hydrolysis 3.6-fold within a new dye, termed SNR700. Leveraging the stabilized NIR absorbance of this dye, we were able to construct the first rhodamine-based, turn-on PAI imaging probe for hypochlorous acid (HOCl) that is compatible with commercial PA instrumentation. This probe, termed SNR700-HOCl, has a limit of detection of 500 nM for HOCl and is capable of producing contrast up to 2.9 cm deep in tissues using PAI. This work provides a new set of rhodamine-based PAI agents as well as a rational design approach to stabilize esterified versions of NR dyes with desirable properties for PAI. In the long term, the reagents described herein could be utilized to enable non-invasive imaging of HOCl in disease-relevant model systems.