A non reactive fluorescent probe capable of tracking mitochondria over a long period of time

The author of this paper: Dr Zhang ruoyao, academician Tang benzhong of Hong Kong University of science and technology and Professor Yu Xiaoqiang of Shandong University published a research paper entitled "reaction free and MMP independent fluent probes for long termitochondria visualization and tracking" on chem SCI A non reactive mitochondrial fluorescence probe capable of tracking mitochondria over a long period of time Work significance and research status: tracking the dynamic changes of mitochondrial morphology and number is of great significance to the research of physiology, pathology and pharmacology Traditional mitochondrial probes based on electrostatic attraction can't track mitochondria for a long time, because these probes will fall off the mitochondria when the mitochondrial membrane potential decreases Although reactive mitochondrial tracking probes are not affected by the changes of mitochondrial membrane potential, these probes will destroy mitochondrial proteins and produce greater toxicity Therefore, it is still a challenge to develop a non reactive, biocompatible and independent mitochondrial tracking probe Design idea: as we all know, most of the cytoplasmic membrane probes, such as DiI and DIO, have long lipophilic fatty chains These probes only rely on the hydrophobic interaction between the long fatty chain and the phospholipid of the cell plasma membrane to dye the cell plasma membrane Inspired by this design idea, and considering that mitochondria also have membrane structure similar to cytoplasmic membrane, the author introduced a long lipophilic fatty chain on the common cationic mitochondrial probe to track mitochondria, as shown in scheme 1 (1c) Once the mitochondrial membrane potential decreases or disappears, the electrostatic interaction will decrease or disappear, but the hydrophobic interaction between LPF and mitochondrial membrane phospholipid will still make the probe fixed on the mitochondria Different from covalent bond, this hydrophobic interaction belongs to non covalent bond interaction, which will not damage mitochondrial protein and make the probe have better biocompatibility (source: chem SCI.) CO staining experiment: next, ecpi-12 and ivpi-12 were co stained with commercial mitochondrial tracking probe (mtdr) respectively The experiment was carried out in normal cells, CCCP treated cells and POM fixed cells The results showed that the co localization coefficients of ecpi-12, ivpi-12 and mtdr were higher under the normal, decreased and disappeared mitochondrial membrane potentials as shown in Fig 1a and B This shows that ecpi-12 and ivpi-12 can achieve specific staining of mitochondria without the influence of mitochondrial membrane potential (source: chem SCI.) mechanism discussion: in order to prove that when the mitochondrial membrane potential is reduced or disappeared, it is the long alkyl chain that makes ecpi-12 / ivpi-12 fixed on the mitochondria, the author synthesized their corresponding short chain molecules ecpi-2 / ivpi-2, and then the short chain molecules were stained and imaged As shown in Fig 1C, in normal living cells, two short chain molecules can stain mitochondria However, when CCCP was added, the pattern of ecpi-2 changed from filiform to punctate, and ivpi-2 entered the nucleus and stained nucleolus The results showed that the two short chain molecules could not stain mitochondria after the mitochondrial membrane potential decreased It should be noted that the only structural difference between ecpi-12 / ivpi-12 and corresponding ecpi-2 / ivpi-2 is the length of the alkyl chain connected to the pyridine end Therefore, the author concludes that ecpi-12 / ivpi-12 can still be fixed on the mitochondria when the mitochondrial membrane potential is reduced due to the effect of long alkyl chain Tracking mitochondrial autophagy: We used ecpi-12 and ivpi-12 to track the process of mitochondrial autophagy HeLa cells were first stained with ecpi-12 / ivpi-12 and lysosomal tracking probe (LTDR), and then induced mitochondrial autophagy with 10 μ m CCCP and 7.5 μ m pepstatin a With the increase of induction time, the fluorescence signals of ecpi-12 / ivpi-12 and LTDR gradually overlapped The co location coefficients of ecpi-12 / ivpi-12 and LTDR increased from 0.15 to 0.81 and 0.25 to 0.82, respectively, as shown in Fig 2 The results show that ecpi-12 and ivpi-12 can track mitochondrial autophagy in situ and in real time (source: chem SCI.) toxicity test: the toxicity of ecpi-12, ivpi-12 and mtdr in HeLa cells can be tested by MTT test As shown in Fig 3, HeLa cells survived more than 80% after incubation with low concentration of ecpi-12 and ivpi-12 for 48 hours However, when HeLa cells were incubated with the same concentration of mtdr for 48 hours, HeLa cell survival rate decreased, especially when the concentration increased The results showed that ecpi-12 and ivpi-12 had lower toxicity and better biocompatibility than mtdr (source: chem SCI.) Summary: in this work, two non reactive fluorescent mitochondrial tracking probes ecpi-12 and ivpi-12 with long alkyl chain were synthesized CO staining experiments showed that ecpi-12 and ivpi-12 could achieve specific staining of mitochondria without the influence of mitochondrial membrane potential Compared with the short chain molecules ecpi-2 and ivpi-2, the long alkyl chain makes ecpi-12 / ivpi-12 still fixed on the mitochondria when the mitochondrial membrane potential is reduced Ecpi-12 and ivpi-12 can track mitochondrial autophagy in situ and in real time, and have low toxicity and good biocompatibility This work provides a new idea for the design of other cell tracking probes The first author of this paper is Dr Zhang ruoyao and Dr Niu Guangle Academician Tang benzhong of Hong Kong University of science and technology and Professor Yu Xiaoqiang of Shandong University are co correspondents The research was supported by NSFC and NSFC of Shandong Province A review of previous reports: academician Tang benzhong of Hong Kong University of science and technology and researcher Wang Shu of the Institute of chemistry of Chinese Academy of Sciences: a series of fluorescent sensor arrays based on AIE molecules are constructed for rapid and accurate identification of pathogenic bacteria adv funct Mater.: academician Tang benzhong of Hong Kong University of science and technology has developed a dual-function AIE material that can be used to monitor and kill multidrug-resistant bacteria Chem SCI.: academician Tang benzhong of Hong Kong University of science and technology proposes a new strategy to find AIE materials from nature Academician Tang benzhong of Hong Kong University of science and technology develops and constructs a new strategy of deep red / near-infrared AIE materials: smaller conjugate system gets longer absorption and emission wavelength Academician Tang benzhong of Hong Kong University of science and technology: aggregation induced emission, AIE)