Application Of Probes Based On CBT-Cys Click Reaction And Supermolecular Self-assembly In Molecular Imaging

Molecular imaging has become an important tool for modern diagnosis and treatment of diseases because it can provide direct biological information of biological entities at the molecular and cellular levels.Based on the CBT-Cys click reaction and supramolecular self-assembly,a small molecule probe,a self-assembled nano-probe and a fluorescent probe were designed and successfully used for sensing biomarkers,imaging self-assembly process,and tracking the tandem assembly and disassembly of nanofibers,respectively.In chapter 2 of this dissertation,based on CBT-Cys click reaction,we developed a silent bioluminescent probe 1,to selectively sense cysteine(Cys)in vitro and image Cys in vivo.The high selectivity of probe 1 for Cys detection lies in the fast conjugate addition of Cys to the acrylate motif of 1 and subsequent intramolecular cyclization to release D-luciferin to generate bioluminescence in the presence of firefly luciferase.We successfully applied probe 1 to selectively detect Cys in the 0-25 ?M linear concentration range with a limit of detection of 88 nM in vitro.Moreover,we also evaluated the capability of probe 1 for sensing Cys in living cells and tumors.We envision that,in the future,probe 1 might be applied to evaluate the Cys roles in more biological processes.In chapter 3 of this dissertation,based on CBT-Cys click reaction,by rational design of an iodinated small molecule NBC-Iod-CBT,we are able to directly observe nanoparticle formation inside cells with nano-CT for the first time.Upon GSH reduction and nitroreducase(NTR)cleavage in vitro,NBC-Iod-CBT subjects to a CBT-Cys click condensation reaction to self-assemble into Iod-CBT-NPs with an average liner absorption coefficient(LAC)value of 0.182±0.078 ?m-1 to x-ray.At hypoxic condition,NTR was found highly expressed in the cytoplasm of HeLa cells.High-performance liquid chromatography analysis and transmission electron microscopy imaging clearly showed Iod-CBT-NPs formation in NBC-Iod-CBT-treated hypoxia HeLa cells.Nano-CT imaging of the cells clearly sho wed the existence of Iod-CBT-NPs in cytoplasm with an average LAC value of 0.172±0.032 ?m-1.Using one type of polymer iodine nanoparticles PMAI as a control,we verified that our method of using LAC to confirm intracellular nanoparticle formation is feasible.We anticipate that our strategy could help people to deeply understand the formation mechanism of intracellular nanostructures in the near future.In chapter 4 of this dissertation,based on supermolecular assembly,we rationally designed a probe PEA-NBD-Yp and employed its fluorescence "on/off" to trace tandem assembly/disassembly of nanofibers in living HeLa cells.In vitro experiments validated that PEA-NBD-Yp could be efficiently dephosphorylated by ALP to yield PEA-NBD-Y which self-assembled into nanofibers with the NBD fluorescence"on".And the PEA-NBD-Y nanofiber was disassembled by GSH,accompanied with fluorescence "off".These ALP-triggered self-assembly and GSH-controlled disassembly with respective fluorescence "on" and "off" were well validated with high-performance liquid chromatography,fluorescence,circular dichroism,and electron microscopy analyses.Living HeLa cell experiments showed that,within 1 h,PEA-NBD-Yp was convert to PEA-NBD-Y by ALP,self-assembling into nanofibers on cell outer membrane with fluorescence "on"(On1).At 8 h,PEA-NBD-Y nanofibers translocated inside cells,together with their "on" fluorescence translocation(On2).At 16 h,PEA-NBD-Y nanofibers were completely disassembled by intracellular GSH,turning the fluorescence "off"(Off2).And the self-assembly/disassembly processes in living cells were validated with the ALP-inhibition/GSH-blocking experiments.We anticipate that our strategy of one probe conferring temporal "on/off" fluorescence signals could provide people with a new tool to deeply understand a biological event(e.g.,microtubulin formation)in living cells in the future.Based on CBT-Cys click reaction and supermolecular self-assembly,we designed a series of probes for molecular imaging.We expect that our strategies could provide new ideas for the detection of disease-related biomarkers,in-depth understanding of the formation mechanism of intracellular nanostructures and biological events in living cells in the future.