Design,Synthesis And Imaging Research Of Fluorescent Probes For Intracellular Microenvironment Detection

The intracellular microenvironment(local environment)includes polarity,viscosity,temperature,hypoxia,and acid-base state(p H)etc,which play indispensable roles in physiological processes.Normal physiological activities are highly dependent on a stable microenvironment.However,the abnormal microenvironment often leads to the occurrence and development of various diseases.Therefore,monitoring the intracellular microenvironment is essential for revealing the pathogenesis of various diseases and their effective diagnosis.Molecular imaging based on fluorescence technology is a non-invasive method that has the advantages of in-situ detection,real-time visualization,and high spatiotemporal resolution,and can monitor subtle changes in the microenvironment of the lesion.Although a large number of microenvironment-sensitive probes have been reported,there are still many problems in the detection of the microenvironment in organisms.First of all,the current detection of the microenvironment mainly limits at the cellular level,and there are few reports on the in vivo level.Secondly,the changing rules of microenvironment in multiple diseases have not been revealed.Moreover,the current superficial research on the microenvironment in the biological system cannot provide effective guidance for clinical medicine.In order to solve the above problems,in this paper,a series of viscosity/polarity sensitive probes are designed and used for the detection of polarity/viscosity in a variety of physiological abnormalities and in vivo models of multiple diseases.The specific content is as follows:(1)A robust fluorescent probe CTPA for potential cancer diagnosis was rationally designed.Owing to its classical donor-?-acceptor(D-?-A)structure,the probe CTPA exhibited outstanding solvatochromism properties.Because of the differences in the polarity and number of lipid droplets(LDs)between cancer cells and normal cells,the probe CTPA fluoresced weakly in normal cells,but strongly in cancer cells.Various types of cancer cells have been successfully distinguished from the normal cells by detecting LDs numbers and polarity.Besides at the cellular level,potential cancer diagnosis has been achieved in living organs and in vivo.Thus,CTPA will be a promising tool for cancer diagnosis and provide a guide for further elucidating biological processes and pathological mechanisms of LDs polarity-related diseases.(2)A new fluorescent probe,CBMC,was designed based on carbazole to diagnose cancer by monitoring the polarity of LDs.Compared with the traditional polarity-sensitive probe,the fluorescence intensity of the probe CBMC showed up-down tendency toward polarity,and the fluorescence of the probe in normal cells was stronger than that in cancer cells.Cancer diagnosis at the cellular and tissue level was realized by the difference in the luminescence of the CBMC in normal cells and cancer cells.In the cancer treatment stage,the tumor area will gradually decrease and the normal area will be relatively enlarged.Therefore,the probe CBMC is more suitable for the evaluation and diagnosis of cancer in the treatment stage.(3)A two-photon(TP)fluorescent probe MND-Lys was designed for sensing lysosomal polarity in multi-disease models.The probe enables quantitative detection of lysosomal polarity in states of cells starvation and dysfunction.By means of the probe,the intrinsic polarity variance at different developmental stages in zebrafish was firstly investigated,and revealed that the polarity of zebrafish in embryo was lower than that in adult zebrafish.Furthermore,the probe MND-Lys was used for the first time to track polarity changes in inflammatory and obese mice.This work not only provides a new platform for monitoring lysosomal polarity in organs and in vivo levels but also contributes potentially to future lysosomal-related diseases diagnosis.(4)An optical agent ERNT was proposed to to investigate the polarity changes during dynamic liver injury.The probe showed excellent solvatochromism and ER targeting,and the changes in ER polarity induced by tunicamycin and dithiothreitol were detected in situ at the cellular level,confirming its potential in monitoring ER polarity in the biological system.Importantly,this work firstly revealed that the ER polarity increases with the evolution of liver injuries.Moreover,the efficacy of hepatoprotective drugs was evaluated successfully by using probe ERNT to detect ER polarity,which confirmed the high clinical application prospect of the probe.(5)A TP fluorescent probe CBI-V with excellent properties was designed for the detection of mitochondrial viscosity in inflammation,fatty liver and tumor lesions.With the probe CBI-V,the change in mitochondrial viscosity caused by monensin or nystatin was monitored in real time.Moreover,the probe is also used for monitoring the viscosity changes of cells,zebrafish and mice under inflammatory pathology.Importantly,the visualization of mitochondrial viscosity was achieved for the first time in fatty liver and tumor models.This work not only reveals the relationship between mitochondrial viscosity and diseases,but also opens up an effective way for the diagnosis of mitochondrial viscosity-related diseases.(6)We proposed viscosity of the tumor microenvironment as a biomarker and further develop a new optical agent,TBM-V,for monitoring the viscosity change of tumor microenvironment so as to realize cancer diagnosis,therapeutic efficacy tracking and anticancer drug screening.When in highly viscous media,near-infrared signals of TBM-V are specifically activated,endowing the probe with the capacity of avoiding biological autofluorescence and achieving high signal-to-noise ratio.The vascular imaging experiment showed showed that the viscosity of tumor blood vessels was greater than that of normal blood vessels,which confirmed the feasibility of tumor microenvironment as a new marker for cancer diagnosis.With the assistance of fluorescence imaging technology,TBM-V enables noninvasive cancer diagnosis and tracking therapeutic outcomes in vivo.In addition,anticancer drug screening was realized by in situ monitoring of tumor microenvironmental viscosity with TBM-V.Furthermore,as a proof of concept,screening of the anticancer drugs is also realized through in situ monitoring of the microenvironmental viscosity fluctuations of the tumor with TBM-V.Notably,the strategies proposed in this work will provide new impetus to current cancer diagnosis and treatment systems.In conclusion,the research work of this paper mainly reveals the change rule of polarity/viscosity in various diseases and elucidates the pathogenesis of diseases.And use the revealed law to realize the diagnosis of related diseases,treatment effect tracking and anticancer drug screening.