Bio-orthogonal Reactions Driven By Sugar Sorting Pathways For The Research Of Stressed Lysosomal Imaging And Antigen-mediated Tumor Immunotherapy

The occurrence of disease is often closely related to the homeostasis of organelles.For example,lysosomes are cellular digestive organs that contain multiple hydrolases that hydrolyze exogenous or endogenous substances.Most lysosomal hydrolases have a terminal mannose-6-phosphate(M6P)group that is transported to the lysosome and functions by the mannose-6-phosphate receptor(M6PR)on the Golgi apparatus.The dynamic changes of lysosomal parameters are closely related to apoptosis and necrosis,autophagy,phagocytic pathogens and neurological diseases.Techniques allowing selective and continuous tracking of dynamic organelles is crucial for modern biology.We herein reported a ship-in-bottle strategy to tag lysosomes using strain-promoted azide-alkyne cycloaddition to couple a pH sensor(RC)with mannose-6-carboxylate(M6C)actively transported into lysosomes via cell sorting.Relative to classical acidotropic sensors prone to dissipate from lysosomes,in situ formed M6C-RC is stably trapped in lysosomes without resort to lysosomal acidity,whereby exhibits "always-on" blue fluorescence indicative of lysosome positioning and red-to-blue fluorescence ratios responsive to lysosomal pH.These advantageous properties enable tracking of stressed lysosomes,discerning distinct lysosomal pH alteration profiles in necrosis over apoptosis.The cell sorting pathway-integrated biorthogonal tagging strategy offers a new route to track stressed organelles with disrupted physiological organelle-probe affinity.With the rising incidence of cancer in the world,finding new ways of treating cancer is of great clinical significance.Prostate cancer is one of the most common malignancies in Europe and the United States.We designed a multifunctional molecule(DNP-DBCOPL)consisting of three parts.One is a prostate-specific antigen ligand(PL)that targets the prostate-specific membrane antigen(PSMA)and the other is 2,4-dinitrophenyl(DNP),which is used for early immunoimaging and later Immunotherapy;third is diphenyl cyclooctyne(DBCO),used for bio-orthorhombic reactions,through the covalent bond of functional molecules attached to the cell membrane surface.This functional molecule has a dual-targeting effect,that is,rapid and stable covalent introduction of an antigen on the cell membrane surface through ligand receptor binding and bioorthogonal reaction,thereby triggering an immune response and killing or inhibiting tumor growth.