Combination of two-photon fluorescent probes for carboxylesterase and ONOO ^- to visualize the transformation of nonalcoholic fatty liver to nonalcoholic steatohepatitis in liver orthotopic imaging

As the most common cause of liver diseases, nonalcoholic fatty liver disease (NAFLD) can be classified into nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH). While NAFL is generally benign, the transition from NAFL to NASH is a cardinal feature of the non-benign liver disease that leads to cirrhosis and cancer, which indicates that tracking the transformation of NAFL to NASH timely is significant for precision management of liver diseases. Therefore, two fluorescent probes (CNFCl and DRNO) have been developed to visualize this pathological event. alpha-Fluorochloroacetamide and alpha-ketoamide was employed as the recognition site for carboxylesterase (CE) in CNFCl and peroxynitrite (ONOO-) in DRNO, respectively. CNFCl (lambda em = 445 nm) and DRNO (lambda em = 560 nm) showed high specificity and sensitivity towards CE and ONOO- respectively. By incubating with CE/ONOO- for 0.5 h respectively, both the emission intensity of CNFCl (linear range: 0-0.2 U/ mL) and DRNO (linear range: 0-17.5 mu M) displayed significant enhancement. As a result, the detection limit of CNFCl and DRNO for CE and ONOO- was calculated as 4.2 mU/L and 0.05 mu M respectively. More importantly, the emission spectra of CNFCl and DRNO in the presence of CE and ONOO- respectively were cross-talk free under the two-photon excitation of 720 nm. This greatly facilitated the simultaneous detection of CE and ONOO- at distinctive channel, thus ensuring the high fidelity of the detection. These two probes were combined to image the fluctuation of CE and ONOO- during the conversion of NAFL to NASH in vitro and in vivo. It was found that while CE displayed a tendency to rise and then reduce during the transition from NAFL to NASH, ONOO- increased continuously, confirming that the combined imaging by CNFCl and DRNO might visualize the transformation of NAFL to NASH. The results provide robust visual tool to decipher the relationship between the stage of NAFLD and the level of CE/ONOO-. We anticipate this study may open new avenues to distinguish NASH from NAFL, which may further promote the study of intracellular biological activities of CE and the development of NAFLD diagnostic methods.