The Synthesis And Applications Of Tumor-targeting And Fluorescence Labelled Gefitinib For Non-small Cell Lung Cancer

Objective:Non-small cell lung cancer(NSCLC)is a highly malignant and poor-prognostic tumor of respiratory system.Systemic administration of anti-tumor drugs is the main method for the advanced patients.During the treatment,the patients will suffer side effects owing to the low selectivity of the drugs will occur,and so will the drug-resistance because of the low concentration.Targeting prodrug is a strategy that the therapeutic payload will be modified as the prodrug with a cleavable linker responding to the internal environment at the lesion and conjugated by the targeting ligand to enhance the selectivity and efficacy,which is more and more used at the field of pharmaceutical industry.Inspired by this,we are engaged to design and synthesize a class of tumor targeting prodrugs to deliver gefitinib to the cancer lesion and monitoring the site of action and the metabolic organs in vitro and in vivo with the help of the conjugated near-infrared fluorophore.In addition,we will access the efficacy of the prodrugs by testing the changes pathological and molecular biological characteristics.We will present a class of fluorescent tumor-targeting prodrugs with a real-time near-infrared fluorescence monitoring in vitro and in vivo.Methods:Two targeting prodrugs(Prodrug Biotin Gefitinib,PBG and Prodrug Polyamine Analogue Gefitinib,PPG)were synthesized which consist of a sulfide bond linking gefitinib as a prodrug and two tumor targeting ligand,biotin and polyamine analogue(PA).On the basis of the two prodrugs,the corresponding fluorescent tumor targeting prodrugs were obtained with conjugating near-infrared fluorophores(azo-BODIPY).For the investigation in vitro,we used gefitinib-sensitive NSCLC cell lines,PC9 cells and gefitinib-resistant NSCLC cell lines,H1650 cells.We used cell viability assays,Annexin-FITC/PI kits and the expression of cleaved-caspase 3 to evaluate the apoptosis-induction of the four prodrugs after the 72-hour administration of the prodrugs.Confocal laser scanning microscope(CLSM)was employed to observe metabolism,elimination,the response of disulfide to glutathione and the transmembrane transport with the intensity or location of fluorescence.Western blot analysis and flow cytometry were employed to examine the changes of the expressions of p-EGFR and p-Akt to detect the molecular mechanisms of the efficacy.The nude mice were injected subcutaneously with PC9 cells and H1650 cells in the armpit,which were used as tumor model for the further research in vivo.The tumor volumes and the body weights were measured during a 2 8-day treatment and at the end of the treatment the tumor masses were stained by immunofluorescence of Ki67,which were used to evaluate the efficacy of the prodrugs.The animal fluorescence imaging system was used to observe the metabolism of every hour after injecting TBG via tail vein.To evaluate the feasibility of the rapid diagnosis and compare with the NSCLC biomarkers,the clinical tissue samples taken out by transbronchial lung biopsy(TBLB)were stained by the fluorescence of carcinoembryonic antigen(CEA)and cytokeratin 19 fragment(CK19)and near-infrared TBG with the detection of flow cytometer.Results:All the compounds were characterized by electrospray ionization-mass spectrometry and 1H and 13C NMR spectroscopy which is consistent with the designed molecules.The cell experiments showed that under the therapeutic concentrations of gefitinib,the four prodrugs inhibited more PC9 cells over gefitinib and only the PA conjugated prodrugs inhibited H1650 cells more than gefitinib rather than biotin conjugated prodrugs.The conjugation of azo-BODIPY hardly affected the efficacy of prodrugs.The results of Western blot analysis indicated that the expression of p-EGFR of H1650 cells downregulated with the treatment of the prodrugs but TBG and PBG hardly suppressed the expression of downstream p-Akt.CLSM showed that TPG and TBG accumulated in the cytoplasm and the transmembrane transportation was energy-dependent.In the experiments in vivo,the differences of body weight between the control group and the therapeutic group were not significant.The PC9 tumor masses could be inhibited by the prodrugs compared with the control group,but the H1650 tumor masses only be inhibited more than gefitinib by TPG and PPG not TBG and PBG.The animal fluorescence imaging showed that TPG accumulated at tumor most and longest compared with any other organs.All the TBLB samples were verified by pathological examinations.The results of flow cytometer indicated that TPG owned the same specificity of CEA and CK19,but TPG owned better sensitivity than that of CEA and CK19.Conclusion:Through conjugated with the tumor-targeted ligand,the four prodrugs can enhance the drug tumor-targeting ability,so as to reduce the side effects and enhance the therapeutic effect.Moreover,PA ligand showed a better inhibitory effect on H1650 cells than biotin,owing to its unique p-Akt inhibitory effect.In addition,near-infrared fluorescence-labeled prodrugs enable real-time and non-invasive monitoring of drug metabolism in vitro and in vivo.This design with enhanced drug targeting ability and real-time detection possibility of drug metabolism will provide new insights into the drug development.