Construction of Fluorescent Probes for Detecting Biologically Active Species and Application in Tumor Phototherapy

Author:Huang Yan

Supervisor:kang zuo chen ling xin


Degree Year:2019





Fluorescent probes have been widely used in analysis and bioimaging because of the good specificity and sensitivity toward the analyte and the non-invasive properties for biological samples.In addition,fluorescent probes can realize non-invasive detection,deep tissue imaging and real-time dynamic imaging for biological samples.The traditional detection methods are difficult to achieve these objectives.Therefore,fluorescence imaging has attracted much more attention in the fields of chemical,biological and medical.In this thesis,a variety of novel small-molecule fluorescent probes and fluorescent nanoprobes were prepared based on organic fluorescent dyes,carbon dots and gold nanorods.The optical properties of organic fluorescent dyes and fluorescent nanomaterials were developed.The small molecule and fluorescent nanoprobes are used to detect some biologically active species.More importantly,we also explore the mechanism of these biologically active species in a variety of diseases,and some nanomaterials further apply to phototherapy of tumors.The main research contents are as follows:1.Imaging of HNO anti-inflammatory effects via a near-infrared fluorescent probe in cell and in rat gouty arthritis modelsNitroxyl(HNO)plays a crucial role in anti-inflammatory effect via inhibition of inflammatory pathways,but the detail of endogenous HNO generation still remains challenging due to the complex biosynthetic pathways,in which the interaction between H2S and NO simultaneously generates HNO and polysulfides(H2Sn)in mitochondria.Moreover,nearly all of the available HNO fluorescent probes are utilized for the imaging of HNO in cells and tissues,instead of the in-situ and in real-time detection of the simultaneous formation process of HNO and H2Sn in mitochondria and animals.Herein,we develop a mitochondria-targeting near-infrared fluorescent probe Mito-JN to detect the generation of HNO in cell and in rat models.The probe is consisted of three moieties:Aza-BODIPY as fluorescent signal transducer,triphenylphosphonium cationic as mitochondria navigator,and diphenylphosphino-benzoyl as HNO-response unit.The response mechanism is based on the aza-ylide intramolecular ester aminolysis reaction with fluorescence emission-on.Mito-JN displays high selectivity and sensitivity towards HNO than other various biologically relevant species.Mito-JN has been successfully applied for the detection of endogenous HNO generation which is derived from the crosstalk between H2S and NO in living cells.The additional generated H2Sn are also verified using our previous probe Cy-Mito.The anti-inflammatory effect of HNO is evaluated in LPS-evoked inflammatory cell models and in rat gouty arthritis models.The result makes our probe be a good candidate for the assessment of HNO-protective effects in inflammatory process.2.Detection of hypochlorous acid fluctuation via a selective near-infrared fluorescent probe in living cells and in vivo under hypoxia stressHypochlorous acid(HOCl),as one of the reactive oxygen species(ROS),plays a crucial role in oxidative damage to tissue proteins in the pathogenesis of various diseases.In spite of the rising interests in HOCl,there only a few biocompatible techniques are available for its direct and non-invasive detection.Moreover,most of the present methods cannot meet the demand of real-time detection on account of the labile and reactive chemical properties of HOCl.Herein,we design a near-infrared fluorescent probe Cy-HOCl for the selective imaging of HOCl in cells and in vivo.Cy-HOCl includes two moieties:a 4-amino-3-nitrophenol group as the response unit;a near-infrared heptamethine cyanine fluorophore as the fluorescent modulator.Cy-HOCl exhibits excellent sensitivity selectivity and sensitivity towards the detection of HOCl.The hypoxia response behavior of Cy-HOCl is evaluated in cells to clarify the relationship between HOCl and hypoxia.The probe is also applied to measurement of HOCl in ex vivo-dissected organs of acute ischemia mouse model,as well as the probe is successfully used for real-time monitoring the changes of HOCl in hypoxic zebrafish model.3.A Simple Method to Synthesize Carbon Dots as Highly Fluorescent Nanoprobe for Detection of Iron Ion and Hypoxia-induced the Change of Ascorbic Acid in Cells and in VivoMaintain the redox balance of biological system is the key factor to ensure the healthy internal environment.Excessive iron ion(Fe3+)accumulate in body can lead to tissue damage,organ failure and eventually death.Fortunately,ascorbic acid(AA)as a reducing agent and has been evaluated for reduction of Fe3+.Moreover,AA plays an important role in decrease hypoxia-induced oxidative stress.Therefore,real-time imaging of Fe3+and AA changes is important for understanding their biofunction in cells.In this part,we develop a highly fluorescent nanoprobe CDs-DB by simple method for the detection of Fe3+and AA.As a representative for the examination,CDs-DB exhibits outstanding sensitivity and selectivity for Fe3+and AA detection.The nanoprobe CDs-DB@Fe is capable for tracing the dynamic changes of AA.Furthermore,CDs-DB@Fe also detects the changes of AA in hypoxic cell model,hypoxia zebrafish model and liver ischemia-induced hypoxia mice model.These all exhibit the decrease of AA under hypoxic conditions due to AA is consumed to neutralize free radicals and decrease hypoxia-induced oxidative stress.The ideal biocompatibility and low toxicity make our nanoprobe has potential for the research of physiological effect of AA in vivo.4.Self-assembly Nanoparticles by Human Serum Albumin and Photosensitizer for Targeted Near-infrared Emission Fluorescence Imaging and Effective Phototherapy of CancerPhotodynamic therapy(PDT)and photothermal therapy(PTT)are effective treatment ways for cancer,and the photosensitizer plays the most important role in the treatment.However,the ideal photosensitizers are insufficient for in vivo tumor treatment.Herein,we develop a small molecule fluorophore Cy-HPT as a novel photosensitizer,which with the advantages of near-infrared(NIR)emission wavelength,high photothermal conversion efficiency and high singlet oxygen generation efficiency.Moreover,a nanoplatform of HSA@Cy-HPT is synthesized by self-assemble of Cy-HPT and human serum albumin(HSA)and Cy-HPT in aqueous solution.Compared to Cy-HPT,HSA@Cy-HPT exhibits not only much stable spectral properties,superior effect of PDT/PTT but also more satisfactory in vivo metabolism.HSA@Cy-HPT shows an outstanding tumor targeting feature in subcutaneous tumor xenograft models due to its enhanced permeability and retention effect for tumor tissue.Furthermore,HSA@Cy-HPT is successfully applied to treatment of tumor xenograft models,the tumor tissue is obviously inhibited without any regrowth and extend survival rate of models.Also,the normal tissue of tumor xenograft models is observed no distinct damage by H&E staining.This work provides a promising therapeutic agent for the synergetic PDT and PTT for cancer.5.An Activatable Near-Infrared Fluorescent Nanoprobe Based on Response of Glutathione for Fluorescence-guided Photodynamic Therapy and Photothermal Therapy of CancerTheranostics nanoplatform offers opportunities for imaging-guided precision therapy and hold great potential for clinical application.In most reported works,the imaging unit is lack of site selectivity,which is always kept“on”modality regardless in normal tissues or tumor sites,increasing the risk of unsafe treatment.Herein,we design a tumor-responsive,near-infrared(NIR)fluorescence-guided theranostics nanoplatform integrating photodynamic therapy(PDT)and photothermal therapy(PTT)functions.A novel NIR fluorescent dyes,CyPT,with excellent optical and PDT properties,is synthesized and linked on gold nanorod(AuNR)to form CyPT-AuNR nanohybrid via a sulfur-sulfur bond that can be broken by glutathione(GSH)with high selectivity and sensitivity.The concentration of GSH is lower in normal tissues than in tumor tissues,the fluorescence of CyPT is on a quenched state by AuNR.By contrast,the high level of GSH at tumor site leads to the break of sulfur-sulfur bond,resulting in the CyPT release and the accomplishment the“off-on”fluorescence response.Followed by precise NIR tumor-imaging diagnosis,the PDT and PTT treatment relying on the released CyPT and AuNR,respectively,can be effectively performed.CyPT-AuNR nanoplatform has been successfully applied to treatment of tumor xenograft models and the nearby normal tissue has been observed no distinct damage.This versatile nanoplatform holds potential for targeted tumor imaging and precision therapy.