Design,Synthesis,and Applications Of Senescence-Associated Enzymes-activated Molecular Probes

Cellular senescence is an irreversible state of cell cycle arrest caused by continuous cell division or in response to various cellular defects.Although non-proliferative,senescent cells are still metabolically active,preserving the potential to secrete a range of a wide range of senescence-associated secretory phenotypes（SASP）（e.g.,pro-inflammatory cytokine,growth factors,chemokines,and matrix degrading enzymes）.These signaling molecules can reprogram the tissue microenvironment and trigger inflammation,which in return result in the senescence of surrounding cells,driving aging and aging-related diseases.Therefore,cellular senescence has become an emerging and important target for the imaging and treatment of aging-related diseases.The development of senescence-targeting molecular tools to monitor the process of cellular senescence,selectively clear senescent cells and regulate the SASP is of great significance to prolong the healthy lifespan and prevent/treat aging-related diseases.In recent years,a variety of senoprobes have been designed and used for the imaging detection and intervention of cellular senescence.However,the paucity of biomarkers limits further advance of senoprobes.Furthermore,most currently available senoprobes are routinely designed by targeting a single senescence-related marker,showing limited sensitivity,specificity,and accuracy.Besides,most currently available senoprobes are not capable of achieving simultaneous detection and intervention of senescent cells.To this end,this thesis aims to find potential biomarkers of cellular senescence and design senescence-associated enzyme-activatable probes for the precise monitoring and intervention of senescent cells.The main contents and results are as follows:（1）Monoamine oxidase A（MAO-A）is a mitochondrial flavoenzyme,which regulates cell oxidative-reduction balance by catalyzing the oxidation of monoamines.Abnormally elevated levels of MAO-A are closely associated with various diseases aging-related diseases such as Parkinson,Alzheimer,and others diseases.Despite high relevance,whether MAO-A can be used as a universal biomarker of cellular senescence is still unknown.Thus,we examined the expression of MAO-A in different senescent cells and demonstrated its potential as a general biomarker of senescence.Then,the senescence-associated MAO-A-activatable probe P_MAO-Awas synthesized and successfully applied for the imaging detection of senescent cells.（2）Design of biomarker-activated senoprobes promises to be a powerful means to improve the specificity during the detection of senescent cells.However,currently available markers are senescence related but not specified,and thus single biomarker activated probes might not be able to accurately differentiate senescence from normal tissue.Thus,we conceived and designed a dual-parameter recognition fluorescence probe(P_βgal-MAO-A)as a general platform for precise imaging of cellular senescence.P_βgal-MAO-Ais composed of a fluorophore and a tandem substrate chain of two enzymes through a self-eliminating linker.Only when theβ-gal and MAO-A are present at the same time could activate the fluorophore,which greatly improved the detection specificity.Moreover,P_βgal-MAO-Acould discriminate SA-MAO-A/SA-β-gal from cancer-associated MAO-A/β-gal.This smart molecular probe shows great potential in the precision imaging detection of senescent cells.（3）In Chapter 3,dual senescence-associated biomarker-responsive senoprobe P_βgal-MAO-Awas designed,synthesized,and successfully applied for the imaging detection of senescent cells.However,upon reaction with the first senescence-parameter,the probes and generated intermediate can diffuse and change location easily in a highly heterogeneous and dynamic biological milieu,resulting in low spatiotemporal selectivity and specificity.Thus,we presented a new strategy for the design of senoprobes via combing receptor-mediated internalization and intracellular enzymatic activation,and constructed a novelβ-galactosidase responsive aptamer-conjugate（Ap-β-gal-Fret）for the precision imaging of senescent cancer cells by targeting two biomarkers of senescent cancer cells（i.e.,senescent surface biomarkers ICAM-1 and intracellular biomarkers senescence-associated beta-galactosidase）.This strategy enabled fluorescence activation only after sequential recognition of two common senescence-associated hallmarks,thereby improving the accuracy and specificity of imaging.More impressively,such sequential recognition design further allowed it to image senescence in three-dimensional multi-cellular tumor spheroids.（4）H₂S has the potential to regulate senescent-associated secretory phenotypes,but its chemical instability and toxicity limit its application as a safe and effective senomorphics.Given the fact that senescent cells show increased lysosomal content and upregulation of SA-β-gal,an activatable and senescence-targeting H₂S prodrug（Lyso-FH₂S-Gal）was designed,synthesized,and applied for visualized regulation of cellular senescence.Lyso-FH₂S-Gal enriched in the lysosome of senescent cells,and reacted with SA-β-gal,resulting in the release of COS and a fluorescent probe.COS was rapidly converted to H₂S by the ubiquitous enzyme carbonic anhydrase（CA）,enabling regulation of the SASP and ROS in senescent cells.The release of fluorophore enabled imaging detection of senescent cells and visualization of H₂S release in real time.Moreover,Lyso-FH_2S-Gal prevented senescence propagation by inhibiting the ASP-mediated crosstalk between senescent cells and surrounding non-senescent cells,further revealing that Lyso-FH₂S-Gal can be a useful platform for the visualized regulation of age-related diseases.