Preparation And Application Of Glutathione-responsive Fluorescent Probe And Nanoprodrug

As a small molecule biothiol,glutathione plays a vital role in many physiological and pathological processes,and its abnormal content is closely related to various diseases.Glutathione is also usually overexpressed in tumor cells,so it may serve as an important biomarker.Thus,constructing a fluorescent probe for detecting and imaging glutathione in cells is of high importance.Furthermore,a theranostic system which can respond to the over-expressed glutathione in tumor region and subsequently release anti-tumor drug would be highly desirable.In this work,we designed and synthesized an intracellular glutathione responsive near-infrared fluorescent probe,in which two molecules of near-infrared fluorophore were connected to each side of a breakable disulfide bridge via a carbonate bond,and the electron-withdrawing action of the carbonate bond quenched the fluorescence of the near-infrared fluorophore.~1H NMR and HR-MS were used to characterize the intermediate products and fluorescent probe.The presence of glutathione cleaved the disulfide bond,activated the near-infrared fluorophore and restored its fluorescence.The spectrometry investigation indicated that,the fluorescent probe exhibited time-dependent and concentration-dependent response to glutathione,with a detection limit as low as 0.27μM.The probe also showed good selectivity towards glutathione.The MTT experiment verified the low cytotoxicity of the fluorescent probe toward cells,and the fluorescence imaging experiments indicated that the probe displayed excellent imaging ability of intracellular glutathione.On this basis,we designed and synthesized a glutathione responsive nanoprodrug.The prodrug,which was prepared by linking a flavonoid-based drug quercetin and a kinase inhibitor gefitinib to each side of the disulfide bridges,could self-assembled into nanoparticles in water media to form a nanoprodrug.Upon administration,the nanoprodrug accumulated in tumor region via the enhanced permeability and retention(EPR)effect.~1H NMR and HR-MS were used to characterize the intermediate products and prodrug.Transmission electron microscopy and dynamic light scattering were used to determine that prodrug could self-assemble into nanoparticles.The presence of glutathione cleaved the disulfide bond and released the two drugs,which promoted tumor cell apoptosis.At the same time,the fluorescence of quercetin was restored to report the drug release.The MTT assays verified the high toxicity of nanoprodrug to tumor cells.Fluorescence imaging experiments indicated that,the nanoprodrug could report the drug release by generating strong intracellular fluorescence in tumor cells.The nanoprodrug exhibted high tumor inhibition efficacy and low side effects in a subcutaneous tumor model of mouse.