Research On Lysosome-targeted Theranostic Molecules Responsive To Senescence-associated β-galactosidase

Senescence is closely associated with a variety of diseases such as atherosclerosis,neurodegenerative diseases,and type 2 diabetes.The accumulation of cellular senescence leads to the aging of the organism and ultimately damages the health of the organism.Therefore,it is important to accurately detect the degree of cellular senescence through scientific means and explore reasonable and effective coping strategies to delay aging to maintain individual health and prevent the occurrence of diseases.During cellular senescence,the levels of senescence-associatedβ-galactosidase（SA-β-gal）,p16,p21 and p53 increase significantly with increasing senescence,so these are often used as biomarkers for senescent cell detection.SA-β-gal,on the other hand,is one of the most classic of the many biomarkers of senescent cells and is widely used in the detection and treatment of senescent cells.Notably,SA-β-gal is a lysosomal-derived exo-glycosidase,encoded by the GLB1 gene,that specifically hydrolyzes theβ-galactoside bonds of different substrates in the lysosome.This enlightens us that if molecules responding to SA-β-gal are targeted to lysosomes,they may provide better diagnostic and therapeutic effects on senescent cells.In addition,fluorescent probes have been reported to achieve targeting of senescent cells mainly throughβ-galactose,and further targeting probes for lysosomes have been little studied.Therefore,this study was based on this design of SA-β-gal-responsive lysosome-targeted senescent cell probes and anti-senescence prodrugs,and the following two parts were carried out:First,a SA-β-gal-responsive lysosome-targeted senescence cell probe Gal-M-D was prepared usingβ-D-galactose as a glycoside and 1,3-dichloro-7-hydroxy-9,9-dimethyl-2（9H）-acridinone（DDAO）as a fluorescent molecule with the introduction of morpholine as a classical lysosome-targeted moiety.The photophysical properties of the probe were determined using UV and fluorescence spectroscopy,and the in vitro response of the probe to SA-β-gal was verified by changes in the fluorescence of the probe after the addition of esterase andβ-galactosidase.Subsequently,senescence models of MDA-MB-231 cells and Hela cells were constructed by doxorubicin and camptothecin induction.The effects of the senescence model construction were confirmed by SA-β-gal staining and fluorescence quantitative PCR experiments.The toxicity of Gal-M-D and the probe Gal-D without the targeting moiety to cells was measured,and the cell survival rate was higher than 70%in the concentration range of 0-50μmol·L^-1,which proved the biosafety of the probe was qualified.In addition,the detection ability of Gal-D and Gal-M-D for senescent cells was compared using the constructed model.Gal-M-D provides better imaging of senescent cells and the modification of the morpholino side chain allows better targeting of the probe to the lysosome as demonstrated by Pearson coefficient calculations.Then,the SA-β-gal-responsive lysosome-targeted anti-senescence prodrugs Gal-M-G and Gal-NC-G were synthesized on this basis.The first anti-senescence prodrug Gal-M-G was prepared usingβ-D-galactose as the glycoside element and anti-senescence gemcitabine as the parent drug,and the lysosome-targeted group morpholine was introduced in the side chain by a click chemistry reaction.The second prodrug,Gal-NC-G,uses dimethylamine as the lysosome-targeted group and additionally modifies the coumarin fluorescent molecule,which can act as a tracer at the same time.In the synthesis of Gal-NC-G,the yield was increased from17.8%to 27.6%by optimizing the click chemistry reaction conditions.Biological experiments of both prodrugs are still in progress.Based on the important value of SA-β-gal in the diagnosis and treatment of senescent cells,and aiming to further improve the targeting of senescent cells,this study constructed the SA-β-gal responsive senescent cell probe Gal-M-D targeting lysosomes,and the SA-β-gal responsive anti-senescent prodrugs Gal-M-G and Gal-NC-G targeting lysosomes.By lysosomal targeting of the senescent cell probe Gal-M-D,it was demonstrated that the introduction of the lysosome-targeted moiety allows for better enrichment of the probe in the lysosome and release of fluorescent molecules in response to SA-β-gal.Also,this study provides a exploration of SA-β-gal responsive lysosome-targeted anti-senescence prodrugs.