A Novel Fluorescent Probe For Studying The Effects Of Reactive Oxygen Species And Heavy Metal Ions On Cell Viscosity

Cell viscosity is an important microenvironment parameter,which plays an important role in maintaining the normal physiological functions of cells,such as signal transduction between biomolecules,interaction between biomolecules,metabolite diffusion,cell growth,apoptosis,autophagy,etc.As an important subcellular organelle,the abnormal viscosity of mitochondria is closely related to many diseases,such as neurodegenerative diseases,atherosclerosis and malignant tumors.In complex cellular environment,mitochondrial viscosity is usually affected by many factors.As active molecules constantly produced in organism,reactive oxygen species（ROS）are closely related to the cell microenvironment,there may be a correlation between the concentration change of ROS and mitochondrial viscosity.In addition,with the aggravation of environmental pollution,the damage of heavy metal ions to human health have received more and more attention.But,there has been no report about effect of excessive heavy metal ions on mitochondrial viscosity.Herein,we designed and synthesized a novel near-infrared fluorescent probe（Mito-NV）for real-time in situ imaging and analysis of mitochondrial viscosity,and used this probe to study the effects of hydrogen peroxide and heavy metal ions on the change of mitochondrial viscosity in living cells.This is of great significance for understanding the execution of mitochondrial functions and the occurrence and development of related diseases.The main content of this paper includes the following two parts:1.We designed and synthesized a novel near-infrared viscosity fluorescent probe（Mito-NV）for real-time in situ monitoring changes of mitochondrial viscosity.Mito-NV was designed based on the twisted intramolecular charge transfer（TICT）principle with using 4-pyridineactonitrile as the electron acceptor and dimethylaniline as the electron donor.The molecular structure of Mito-NV was characterized by ¹H NMR,¹³C NMR,and high-resolution mass spectrometry.In a low-viscosity environment,the intramolecular single bond rotates at a high speed,and the fluorescence signal of Mito-NV is weak;while in a high-viscosity environment,the rotation of the intramolecular single bond is inhibited,and Mito-NV emits strong near-infrared（685 nm） fluorescence signal.Chemical analysis shows that Mito-NV has a good linear response to viscosity without affected by polarity,pH and various components contained in cells.Cell experiments show that Mito-NV has low cytotoxicity,good photostability,and can effectively target mitochondria.Under the induction of starvation and stimulation with drugs（nystatin,rotenone）,Mito-NV can give a good indication on changes of mitochondrial viscosity.In short,Mito-NV has high sensitivity and good selectivity towards viscosity,which provides a good tool for studying the influence of various factors on cell mitochondrial viscosity.2.We used Mito-NV to explore the effects of hydrogen peroxide and heavy metal ions on the viscosity of mitochondria.First,after exogenous H₂O₂ and endogenous H₂O₂（PMA）stimulating HepG2 cells,Mito-NV was used to visualize and image the mitochondrial viscosity.The results showed that the viscosity of mitochondria increased significantly after incubating with H₂O₂ and PMA.After incubating with heavy metal ions(Cu²⁺,Co²⁺,Cd²⁺),we also used Mito-NV to visualize and image mitochondrial viscosity.The results showed that the viscosity of mitochondria decreased after induction of heavy metal ions(Cu²⁺,Co²⁺,Cd²⁺).The above results indicate that hydrogen peroxide and heavy metal ions have different effects on mitochondrial viscosity.This provides an experimental method for further research on the effects of other factors on mitochondrial viscosity,and provides a possible tool for studying the mechanism of occurrence and development of related diseases.