Functional Fluorescent Nanoprobes For Imaging Analysis Of Active Molecules In Living Cells And In Vivo

Intracellular active molecules,including active small molecules and macromolecules,are the regulatory network of the organism and the basis of all life activities.The active small molecules,including reactive oxygen species（ROS）,metal ions,H⁺and so on,play a vital role in the signal transduction of cells.They are closely linked to the physiological and pathological processes,aging of organisms,the occurrence and development of various diseases.The active macromolecules,including deoxyribonucleic acid（DNA）,ribonucleic acid（microRNA,tRNA,mRNA）and protein,play an important role in biological growth,development,inflammation and cancer.More and more studies show that various active molecules in cells and organelles are in interaction and mutual cooperation with each other.The active molecules regulate the cell function and are closely related to the occurrence and development of major diseases.For instance,hydrogen peroxide（H₂O₂）and pH fluctuations are closely correlated with mitochondrial dysfunctions,which are implicated in various human diseases including neurodegenerative disorders and cancers.It has been found that the variations of pH and O₂^·-were commonly accompanied with autophagy and apoptosis.In addition,abnormal changes of microRNA（miRNA）and H₂O₂ present concurrently in cancer,ischemia-reperfusion injury,and cardiovascular diseases.Therefore,developing effective methods for real-time monitoring the changes of active molecules in cells and in vivo is of great significance in revealing their interactions in various physiological and pathologic processes.Fluorescent detection method and imaging technology provide powerful tools for real-time and accurately detecting active molecules due to their high sensitivity and temporal resolution.Fluorescent nanoprobe has many advantages,such as good biocompatibility,good solubility,etc.,which has aroused great concern in the field of analytical chemistry,biology and medicine.In particular,fluorescent nanoprobe has been widely used in the detection of various active molecules,which effectively promoted further understanding the role of active molecules in the occurrence and development of the disease.However,most of the methods are specific to only one kind of active molecule rather than multiple active molecules,which greatly hindered in-depth study on the collective effects of multiple active molecules in the pathological processes.Therefore,developing a single probe for simultaneously detecting a variety of active small molecules and macromolecules,will provide important ways for studying the interactions of intracellular active molecules in regulating of cellular functions.In this paper,a series of novel fluorescence nanoprobes were designed and synthesized to simultaneously detect and image mitochondrial pH/H₂O₂ and O₂^·-/pH,visualize the changes of mi RNA/m RNA and H₂O₂/miRNA in living cells and in vivo,and further explore their relationship in alcoholic liver disease and heart ischemia-reperfusion injury.It mainly includes the following sections:1.A novel mitochondria-targeted fluorescent nanosensor was developed for real-time imaging of the fluctuations of H₂O₂ and pH in living cells.The fluorescence probes for detecting pH and H₂O₂ were loaded in the small-sized mesoporous silica nanoparticles（MSN）.Then the polyethylenimine was attached to cap the pores of MSN,and the triphenylphosphonium was further modified to target mitochondria in living cells.The nanosensor could effectively target to mitochondria and successfully achieve real-time imaging of mitochondrial H₂O₂ and pH fluctuations in living cells.Notably,the developed nanoprobe can provide a promising tool to further investigate the interplaying roles of multiple organelles.2.A dual-ratiometric fluorescent nanoprobe was developed for quantitatively differentiating the dynamic process of O₂^·-and pH changes in autophagy and apoptosis in HeLa cells.Rhodamine B loaded mesoporous silica core was used as the reference,and fluorescent probes for pH and O₂^·-measurement were doped in the outer layer shell of SiO₂.Then,chitosan and triphenylphosphonium were modified on the surface of the SiO₂.The kinetics data revealed that the changes of pH and O₂^·-during autophagy and apoptosis in He La cells were significantly different.The pH value was decreased at the early stage of apoptosis and autophagy,whereas the O₂^·-level was enhanced at the early stage of apoptosis and almost unchanged at the initial stage of autophagy.At the late stage of apoptosis and autophagy,the concentration of O₂^·-was increased.While the pH was decreased at the late stage of autophagy and almost unchanged at the late stage of apoptosis.We hope that the present results provide useful information for studying the effects of O₂^·-and pH in autophagy and apoptosis in various pathological conditions and diseases.3.The nanoprobe is designed by decorating the molecular beacons（MBs）onto the surface of gold nanoparticles（AuNPs）.Typically,the fluorescence signal of fluorophores linked with the MBs was efficiently quenched by AuNPs.Whereafter,the corresponding MBs were opened to produce fluorescence signals after interacting with the miR-155 and OPN mRNA,which are biomarkers of alcoholic-induced fatty liver and steatohepatitis,respectively.The experimental results indicate that the nanoprobe can effectively differentiate alcohol-induced fatty liver and steatohepatitis.Moreover,the nanoprobe can monitor the transmutation process of alcohol-induced fatty liver to steatohepatitis and assess the remission effects of N-acetyl cysteine for alcohol-induced liver injury,which provides new ways for early warning,treatments,and prognosis of ALD.4.A novel crown-like silica@polydopamine-DNA-Ce O₂ nanocomposite was developed by assembly of silica@polydopamine-DNA1 nanoparticles decorated with satellite CeO₂-DNA2 nanoparticles for the detecting and imaging of microRNA-21（miRNA-21）and H₂O₂ in simulated ischemia-reperfusion（IR）injuryin living cells and in vivo.The miRNA-21 was found to be regulated by H₂O₂ via PI3K/AKT signaling pathways in H9C2 cells in simulated ischemia-reperfusion injury for the first time.H₂O₂ and miRNA-21 are over produced during mimicked heart ischemia-reperfusion injury,suggesting that they are closely related to reperfusion injury.All these results reveal that there is definite cross-talk between mi RNA-21 and H₂O₂ in IR injury.The current method can provide a promising strategy to further explore the interplaying roles between ROS and miRNAs in other pathological processes.