Peroxynitrite Activatable Double Spiral Ring Derived Fluorescent Probe For Imaging Of Drug-induced Liver Injury

Objective:The liver is the main organ for the metabolism of endogenous and exogenous substances in the body and is therefore more susceptible to drug-induced damage than other organs.Free radicals,ROS,and RNS are major by-products of drug metabolism in the liver and can lead to apoptosis and acute liver injury.RNS and ROS can be used as biomarkers to predict drug-related liver injury,such as ONOO^-.Therefore,it is crucial to develop a simple,reliable and accurate technique to monitor intracellular ONOO^-change.Method:Fluorescein and rhodamine were combined with spirobenzopyran to obtain the fluorescent probes FLSP and Rhod-SP,respectively.Subsequently,we characterized its chemical structure using HR-MS,¹H-NMR and ¹³C-NMR.Some common biological interferents were selected to study the specificity of the probe for ONOO^-.Evaluation of the sensitivity of FLSP response to ONOO^-using fluorescence titration.The reaction stability of the probe FLSP was investigated in different p H conditions and solvents.MTT experiments were chosen to investigate the biocompatibility of the probe FLSP.Endogenous,exogenous and APAP-stimulated production of ONOO^-in Hep G2 cells and zebrafish were imaged using laser confocal microscopy.Finally,changes in the concentration of ONOO^-in mouse leg inflammation and drug-induced liver injury were imaged by the Ami HT in vivo imaging system.Results:HR-MS,¹H-NMR and ¹³C-NMR results indicated the successful synthesis of probes FLSP and Rhod-SP.Fluorescence titration results showed that the fluorescence intensity at 535 nm increased 22.5-fold before and after the reaction of probe FLSP with ONOO^-,and the detection limit was as low as 122 nm.Probe FLSP showed higher sensitivity to ONOO^-compared to probe Rhod-SP.The results of photostability and specificity experiments showed that the response performance of probe FLSP to ONOO^-was not affected by p H,polarity and other interferents commonly found in vivo,and had good photostability and specificity.MTT experiments showed that the survival rate was higher than 85%even when Hep G2 cells were incubated with 12mM FLSP for 24 hours.Cells and zebrafish experiments showed a significant fluorescence enhancement after administration of SIN-1（an ONOO^-exogenous donor）,LPS and APAP compared to the FLSP-only incubation group.Mouse experiments showed significant fluorescence enhancement in the legs and liver of mice given LPS and APAP compared to the saline group,while the liver fluorescence intensity was again significantly reduced in UDCA,NAC and GSH treated mice.Conclusion:Two fluorescent probes FLSP and Rhod-SP were successfully synthesized in response to ONOO^-.The fluorescent probe FLSP with high sensitivity and low detection limit was screened based on fluorescence titration results.comprehensive fluorescence spectroscopy experiments showed that the probe FLSP exhibited excellent specificity,sensitivity and response stability to ONOO^-.The probe FLSP can be successfully applied to endogenous and exogenous ONOO^-detection in Hep G2 cells and zebrafish,and confirmed the activation of CYP450-mediated ONOO^-production during APAP-induced DILI.In addition,in vivo imaging using FLSP revealed ONOO^-overexpression at the site of inflammation and in the damaged liver.Consistent liver fluorescence imaging and H&E staining results confirmed the repairing role of UDCA,NAC and GSH in vivo.These results suggest that FLSP has great potential in the early diagnosis of DILI and provides a new approach for drug screening.