Ratiometric Fluorescent Probes For Lysosomal PH Measurement Based On Spiropyran Molecular Switches And Their Biological Application

Lysosome is an important subcellular organelle in cells.The range of lysosome pH is from 4.5 to 5.5.Due to its acidic environment,lysosome plays an important role in intracellular lysosomal metabolism,cell cycle,cell autophagy,cell membrane self-cleaning and apoptosis.However,the recent studies suggest the appearance of lysosomes in cancer cells is different from those in normal cells,which are larger and more irregular.And the release of cathepsins from cancer-cell lysosomes into the extracellular space can promote tumour progression,which makes lysosome become an anticancer target.Therefore,the clear visualization of lysosomes and detection of pH can not only be conducive to comprehend the physiological functions of lysosomes,but also provide new strategy for the diagnosis and treatment of cancer.Currently,some excellent lysosome-targeted fluorescent probes for detecting lysosome pH have been reported owing to their handy operation,noninvasiveness,and real-time characteristics.Among reported fluorescence probes,it is generally known that ratiometric fluorescence probes can avoid outside interference through the self-calibration of two emission bands to obtain more accurate measurements,and thus the ratiometric probes for detecting lysosome pH has become research hotspots in this field.Generally,spiropyran fluorescent probes are used as photodynamic sensors.However,in addition to light irradiation,pH change can also regulate the isomeration between the open form and the closed form.Under suitable acidic conditions,the C_spiro-O bond of spiropyran is easily protonated,forming an-conjugated open form and further changing the fluorescent signal.On account of this principle,ratiometric lysosomal pH fluorescent probes HAN1,HAN2,HAN3 and HAN4 based on coumarin-spiropyran structure were designed and synthesized in this article.The pH characteristics,interferences and light stabilities of the probes were systematically studied.The results showed that the open forms of the four probes could occur the pH range of 4.0-7.0,accompanied with fluorescence changes from blue to red without the influence of negative ions,cations and light.HAN1 and HAN3 were also applied to the imaging study of the increase of lysosomal pH induced by chloroquine.The results showed that the intensity of blue fluorescence enhanced and the intensity synchronously weakened in the red channel.Hence,HAN1 and HAN3 successfully realized the ratiometric detection of pH and real-time monitoring the variation of pH in lysosomes.At the same time,these probes were also applied to monitor the pH changes of lysosome before and after thermal injury.The fluorescence imaging result also shows the fluorescence intensity of the blue channel enhanced and the fluorescence intensity of the red channel synchronously weakened,which indicated that lysosomal pH gradually increased in the wake of the increase of cell temperature.Herein,This design method based on the spiropyran switches can provide a new strategy for the development of ratiometric lysosome pH fluorescence probe.Meanwhile the ratiometric probes also provide more accurate information for cancer detection and pathogenesis research.Herein,a novel series of real-time and accurate fluorescent probes for detecting lysosome activity in living cells have been developed and a new design strategy for the ratiometric lysosome pH fluorescent probe has been provided.