Development Of GGT-activable Near-infrared Fluorescent Probes For In Vivo Imaging

Molecular imaging probes that allow for non-invasive visualization and measurement of biomolecules of interest in living systems have played important roles in precise diagnosis of diseases.It is highly disireable to develop molecular imaging probes capable of real-time and non-invasive detection of biological targets in vivo with high sensitivity and specificity.y-Glutamyl transpeptidase(GGT),is an important amino transpeptidase modulating intracellular redox metabolism,and is closely related to the tumor growth,migration and drug resistance.It is recongnized that GGT activity is significantly higher in many maglinant tumor cells,and the aberrant expression of GGT can be considered a potential biomarker for cancers.As such,development of molecular imaging probes amenable to detect GGT activity in vivo can help to early diagnose diseases and differentiate benign and maglinant tumors.Recently,there have been many optical imaging probes have been actively developed for the detection of GGT in tumor cells.However,the applications of these probes for in vivo imaging of GGT activity are remaining challenging due to two limitations:1)lack of near-infrared(NIR)fluorescence that ensures deep tissue penetration depth and high in vivo sensitivity;2)lack of tumor-targeting delivery capability that allows to specifically detect GGT activity in tumor tissues after systemically injection into living animals.This dissertation focuses on the development of novel GGT-activatable NIR fluorescent probes for non-invasive and real-time detection of GGT activity in the tumors of living mice.The main contents include two parts:1.Development of activatable NIR probe for fluorescence imaging of GGT in tumor cellsA new GGT-activatable NIR fluorescent imaging probe(GANP)is developed by linking a GGT-recognitive substrate y-glutamate(y-Glu)and a NIR merocyanine fluorophore(mCy-Cl)with a self-immolative linker p-aminobenzyl alcohol(PABA).We demonstrate that GANP is stable under physiological conditions,but can be efficiently activated by GGT to generate～100-fold enhanced fluorescence at 720 nm,offering high sensitivity(detection limit:～3.6 mU/L)and specificity for the detection of GGT activity.Fluroescence imaging results show that the membrane-bound GGT in live tumor cells can rapidly recognize GANP and relase fluorescent and hydrophobic mCy-Cl capble of entering into tumor cells and localizing mainly in lysosomes,resulting in bright intracellular fluorescence for real-time detection of endogenous GGT activity as well as rapid evaluation of the inhibition efficacy of GGT inhibitors in living tumor cells.Notably,the deep tissue penetration ability of NIR fluorescence at 720 nm can further allow GANP to detect GGT activity in xenograft tumors following intraturmoal injection into mice.2.Targeted delivery of GGT activatable NIR fluorescent probe for in vivo imaging of tumorsIn part two,we have developed a tumor-targeting and GGT activatable NIR fluorescent probe(c-RGD-GANP)through introducing an αvβ3-integrin-receptor-targetable ligand,c-RGD into GANP,aiming to improve GANP’s aqueous solubitility and in vivo imaging specificity for tumors.We demonstrate that c-RGD-GANP is highly soluble and fluorescence off in aqueous solution,but can be remarkably activated by GGT,resulting in-230-fold significant turn-on fluorescence at 712 nm capable of detecting GGT with high sensitivity(detection limit:～2.9 mU/L).Cell studies show that c-RGD-GANP can be specifically cleaved by GGT,and the cleaved fluorescent product can selectively enter into U87MG tumor cells via αvβ3-receptor-mediated endocytosis.As such,remarkably enhanced intracellular NIR fluorescence was observed in the tumor cells only,allowing to differentiate the tumor cells from the GGT-positive,αvβ3-deficient normal cells(e.g.,HEK293 cells).Morevoer,c-RGD-GANP is successfully applied for the real-time and noni-nvasive detection of GGT activity in xenograft U87MG tumors following i.v.administration.This study reveals the advantage of using a combination of receptor-mediated tumor cell uptake and GGT-triggered activation to design fluorescence probes for improved cancer imaging specicity in vivo,which could facilitate precise cancer diagnoses.