Construction Of Activatable Xanthene-based Probes And Their Applications In Liver Diseases’ Theranostics

Fluorescence imaging and optoacoustic imaging are two noninvasive detecting methods.Especially optoacoustic imaging,which is a hybrid imaging mode that integrate optics and acoustics,can partially overcome drawbacks of strong tissue scattering and low tissue penetration depth from optical imaging,thus achieving higher imaging resolution and contrast.The liver is one of the most important metabolic organs in the human body.Because of frequent exposure to toxic materials,the liver is susceptible to injury.The loss of hepatocyte function is often accompanied by abnormal expression of specific biomarkers.In-situ detection and imaging of liver diseases-related biomarkers utilizing activatable probes is of great significance for precision and early diagnosis in liver diseases.In addition,theranostics is committed to the synchronous combination of disease diagnosis and therapy,so as to achieve accurate diagnosis and therapy in the whole process and bring a better quality of life to patients.However,most developed probes are not theranostic platforms,and few of them can achieve high-quality fluorescence/optoacoustic dual-mode imaging.Three kinds of activatable xanthene-based probes have been constructed and successfully applied in liver diseases theranostics.（1）Design and preparation of H₂S-responsive xanthene-based molecular probe NR-NO₂.Metformin is the most prescribed oral drug in diabetes treatment,long term or overdose of which can cause serious liver injury,which is closely related to the excessive production of H₂S in the liver.Therefore,monitoring the expression of hepatic H₂S would be an effective method to evaluate the hepatotoxicity of metformin.Herein,a H₂S-responsive xanthene-based molecular probe NR-NO₂ is constructed,in which the benzothiazole-xanthene basic skeleton is used as the near-infrared chromophore,and the dinitrobenzene ether modified on xanthene serves as the H₂S-responsive group and the fluorescence quenching group.After the specific reaction with H₂S,NR-NO₂ will be transformed into the activated probe NR-OH,whereby the strong electron acceptor（dinitrobenzene ether）is replaced with the strong electron donor（hydroxyl）,so as to generate strong fluorescence emission at 700-760 nm and strong optoacoustic signal.This xanthene-base molecular probe NR-NO₂ is applied to specifically detect the upregulation of hepatic H₂S,thus achieves imaging and diagnosis of metformin-induced liver injury,also evaluates the severity of liver injury in a noninvasive way.（2）Design and preparation of NO-responsive xanthene-based nanoprobe.Chinese herbal medicine is widely used all over the world and has a long history.Studies have shown that the improper use of Polygonum multiflorum（PM）,a common Chinesed herbal medicine,can induce liver injury,which is closely related to the excessive production of reactive nitrogen species（such as NO）in liver region.Therefore,monitoring the expression of hepatic NO is an effective method to evaluate the hepatotoxicity of PM.Therefore,a NO-responsive xanthene-based molecular probe IX-2NH₂ is constructed,in which the indole-xanthene basic skeleton is used as the near-infrared chromophore,and the o-aryldiamine modified on xanthene serves as the NO-responsive group.After the specific reaction with NO,IX-2NH₂will be transformed into the activated form（IX-TAZ）,whereby the strong electron donor（o-aryldiamine）is replaced with the weak electron donor（triazole）,thus quenching the fluorescence emission at 690-760 nm and the optoacoustic signal.The molecular probe IX-2NH₂ and the conjugated polymer DPP-TT based on diketopyrrolopyrrole are encapsulated by choleric acid modified phospholipid DSPE-PEG₂₀₀₀-Ch A to form the NO-responsive nanoprobe,which can realize the ratio optoacoustic imaging of hepatic NO.DSPE-PEG₂₀₀₀-Ch A not only endows the nanoprobe with liver-targeting-imaging capability,but also improves its biocompatibility.This NO-responsive xanthene-based nanoprobe is applied to image and diagnose PM-induced liver injury in a noninvasive way by real-time monitoring the heptic NO levels.（3）Design and preparation of H₂O₂-responsive xanthene-based nanoprodrug BH-EGCG&NAC@MM.Autoimmune hepatitis is one of the most complex liver diseases in clinical.Excessive production of reactive oxygen species（such as H₂O₂）in inflammatory liver can be used as the biomarker in diagnostic imaging and utilized as the trigger for in-situ on-demand release of anti-inflammatory drugs.Firstly,a H₂O₂-responsive molecular prodrug BH-EGCG is constructed,in which the boric acid group modified on the xanthene-based chromophore is coupled with the ortho dihydroxy group on natural polyohenol EGCG,and the formed borate ester bond is used as the specific response site of H₂O₂.BH-EGCG is an amphiphilic molecule,which can readily form stable nanoparticles in aqueous medium.Then,both BH-EGCG（the molecular prodrug）and NAC（the ROS scavenger）are coated with macrophage membrane to obtain nanoprodrug BH-EGCG&NAC@MM,which is applied in theranostics of concanavalin A-induced autoimmune hepatitis and carrageenan-induced hind paw edema.Benefiting from the inflammation-homing effect of the macrophage membrane,BH-EGCG&NAC@MM can migrate to inflammatory lesions and specifically response to over-expressed H₂O₂ in the inflammatory microenvironment.After being activated with H₂O₂,the molecular prodrug BH-EGCG can be transformed into the activated chromophore BH-OH,so as to generate obviously enhanced fluorescent emission at 700-750 nm and optoacoutic signals,which is utilized in diagnosis of inflammation or tracking therapeutic efficacy.Meanwhile,the released EGCG and NAC can effectively inhibit NF-κB pathway and suppress the activation of NLRP3 inflammasome,thus give out significant anti-inflammation therapeutic effect.