Investigation Of Enzyme-responsive Cyanine-based Fluorescent Molecular Probes Towards Their Bioimaging Applications

Due to molecular imaging probes can significantly improve the contrast between normal tissues and diseased tissues(tumor or inflammatory areas),a lot of research works have been carried out on developing more functional and effective molecular probes.In the process of cells become cancerous or inflamed,changes in cell metabolism often occur first,including the abnormal expression of some proteases.Although CT,MRI,and nuclide imaging(PET/SPECT)all have their own imaging advantages,fluorescence imaging has its unique benefits of high sensitivity,specificity,and real-time imaging in living cells comparing to other image techniques.Especially with the development of clinical imaging equipment,optical imaging has been playing an increasingly prominent advantage in intraoperative navigation.Based on the advantages of optical imaging,this research paper focuses on the design of three organic small molecule probes with response to the abnormal expression of related enzymes in inflammation and tumors.The main contents include the following three aspects:First,Previous study showed that pyroglutamate aminopeptidase 1(PGP-1)has a relationship with the immune response in cells.However,whether PGP-1 is involved in inflammatory response in vivo and can serve as a new inflammatory cytokine are still unclear.To address these issues,we develop a new near-infrared“off-on”fluorescent probe with hemicyanine as fluorophore and L-pyroglutamic acid as recognition group,which exhibits high selectivity and super sensitivity(reaction time:26 min,detection limit:0.18 ng/m L).The Michaelis constant(K_m)of the as-synthesized probe for PGP-1 were determined to be 7?M,which is much lower than that of commercial probe(K_m,360?M),indicating the as-synthesized probe has stronger affinity for PGP-1.Moreover,the probe shows good biocompatibility examined by standard MTT assays.With this probe,the up-regulation of PGP-1(evidenced by western blot)in BALB/c mice legs and livers under the stimulation of two main immunopotentiators is revealed for first time.The occurrence of inflammatory process(including tissue necrosis)in mice is also determined by up-regulation of tumor necrosis factor-?and hematoxylin-eosin staining.Interestingly,it is revealed for first time that knocking down PGP-1 with small interfering RNA(si RNA)leads to the weakness of inflammatory process in RAW264.7 cells.These important findings as well as the previous results suggest that PGP-1 is indeed involved in body's immune response and can be a new inflammatory cytokine.Second,we firstly develop a new spectroscopic off-on probe with high sensitivity(reaction time:14 min;detection limit:5.8 ng/m L)and good selectivity for fluorescence imaging the hypoxic status of tumor cells via its enzymatic reaction with nitroreductase in vitro and in vivo in the presence of dimethyl sulfoxide.Inspired by the recent investigations on metal-organic frameworks(MOFs),a dual p H and ATP responsive ZIF-90 nano-platform was synthesized,and then PEG was post-modified through Schiff base reaction.This allows the platform as a carrier to load hypoxia-responsive probe to investigate its response to enzyme in cell and in mice without using dimethyl sulfoxide as co-solvent.Consequently,the two probes we synthesized here can successfully respond to nitroreductase for turn-on fluorescence imaging in cellular level and in tumor-bearing mice.Notably,probe 2 can efficiently accumulate in tumors after long term ciculation and passive targeting via tail vein injection This is the first time that an enzyme-responsive organic small molecule probe has been mounted on one of the MOFs which will open up ways to the application of both organic probes and MOFs.Third,we designed and synthesized a small molecule organic probe which owns specific response to?-glutamyl transpeptidase by using hemicyanine as the fluorophore and glutamic acid as the recognition group.In vitro characterization experiment,results show that the synthesized probe has the ability to respond to?-glutamyl transpeptidase,and the detection limit of the probe for?-glutamyl transpeptidase is 4.8 U/L,which is lower than normal level of serum.MTT experiments show that the as-synthesized probe has excellent biological safety,so it can be used in cellelar level imaging and in vivo imaging;immunoblotting results show that both KYSE 30 and KYSE 510 esophageal squamous cancer cells have a high level expression of?-glutamyl transpeptidase.Good laser confocal imaging results were obtained through incubation of probe with KYSE30 and KYSE 510.Moreover,the specific response of probe to?-glutamyl transpeptidase was verified by si RNA transfection experiments,inhibitor experiments,and western blot experiments of the cells.