Construction And Bioimaging Study Of Fluorescent Probes And Fluorescent Dyes Based On Pyronin Dye Platform

Recently,due to the high sensitivity,visualization,no radiation,real-time imaging ability,fluorescent imaging technology is widely applied in the fields of molecular biology,physiology,clinical diagnostics and oncology,which has become one of the important technology in biological science filed.Cancer is a disease in which cells grow and divide out of control,and the death rate increases year by year.Currently,the common methods for tumor diagnosis include single-photon emission computed tomography,X-ray imaging,magnetic resonance imaging,positron emission tomography etc.These technologies do not often work well until the middle to late stages,when metastasis and diffusion have occurred,due to the lack of sufficient specificity and sensitivity.By comparison,fluorescence probe-based imaging technique offers the exciting opportunity to overcome these limitations.To enhance the specificity,in addition to utilizing the conventional enhanced permeation and retention(EPR)effect,the most common strategy for such probes is chemical conjugation of fluorophore with targeting ligands including chemical molecules,peptides,proteins,antibodies,and aptamers,which could specifically bind to biomarkers overexpressed in cancer cells.However,although fluorescence imaging diagnosis of the differences between cancer cells and normal cells by such probes is useful to classify patients towards personalized therapy,using the differences to diagnose a wide range of cancers is often not possible due to the genetic or phenotypic heterogeneity of cancer cells.What is more,lysosomes have a strong correlation to the tumor invasion and progression,the development of fluorescent probes that can noninvasively and specifically image and track lysosomes would be very significant for studying lysosomal trafficking in cancer and decreases.To date,there are two basic types of lysososmal probes whose lysosomal localization correlates with endocytosis pathway and lysosomal acidity,respectively.Regrettably,due to the energy-dependency,the endocytosis process often requires hours to days,and once lysosomal pH increases in response to stimulation,the latters probes may leak out from lysosomes,leading to the decreased fluorescence signal or nonspecific staining which partially compromises their use in real-time tracking of lysosomal migration,morphological change,and trafficking.In this thesis,based on pyronin fluorophores bearing excellent photophysical properties,biocompatibility and easy to control by fluorescent sensing mechanisms,we have designed and synthesized a series of probes,which in order to be applied to diagnosis of cancer cells from normal cell,selectively and sensitively image cancer-related biological small molecule or cell organelle bioimaging research.For this purpose,we have carried out the following words:In Chapter 2,a 2?(diphenylphosphino)phenol-functionalized pyronin POP was presented which could efficiently penetrate cancer cell,rather than normal cell membranes by active transport of organic-anion transporting polypeptide(OATP)transporters overexpressed in many types of cancer cells,and then is activated by intracellular cysteine(Cys)and glutathione(GSH)to produce green-emission aminopyronin NP and red-emission thiopyronin SP,thereby enabling its use in dual-channel fluorescence diagnosis of a wide range of cancer cells with excellent contrast.Crucially,POP also displays the ability of dual-channel fluorescence diagnosis of cancer tissues from tumour xenograft models of mice and harvested surgical specimens of patients,thus holding great potential for the clinical applications.In Chapter 3,we brought forward a Si-pyronin-based fluorescent probe,named PSiR,which could selectively and sensitively image the pathologically more relavent highly reactive oxygen species(hROS:HClO,HO~?,and ONOO~-)in lysosomes of cancer cells.Compared with many of the existing hROS fluorescent probes,its superiorities are mainly embodied in the high stability against autoxidation and photoxidation,near-infrared exitation and emission,fast fluorescence off-on response,and specific lysosomal localization.Its practicality has been demonstrated by the real-time imaging of hROS generation in lysosomes of human non-small-cell lung cancer cells stimulated by anticancer drug?-lapachone.Moreover,the probe was sensitive enough for basal hROS in cancer cells,allowing its further imaging applications to discriminate not only cancer cells from normal cells,but also tumors from healthy tissues.Overall,our results strongly indicated that PSiR is a very promising imaging tool for the studies of ROS-related LCD of cancer cells,screening of new anticancer drugs,and early diagnosis of cancers.In Chapter 4,a new class of lysosome-targeted fluorescent dyes,termed amino-Si-Pyronin(ASiRs),were developed based on Si-pyromin dyes,which possess the intrinsic lysosome-targeted ability and long-term retention ability in lysosomes that is independent of lysosomal acidity and endocytosis pathway.As a result,ASiRs show stable lysosomal localization against lysosomal pH changes.These merits,coupled with their excellent two-photon photophysical properties,large stokes shift,excellent cell-membrane-permeability and negligible cytotoxicity,make ASiRs very suitable for real-time and long-term tracking of lysosomes in living cells or tissues without interference to normal cellular processes.ASiRs provides an important platform for studying the physiological roles of lysosomes,which are closely related to the occurrence and development of cancer.