Intracellular Self-Assembly Of Ru?bpy?₃²⁺ Nanoparticles Enables Persistent Phosphorescence Imaging Of Tumors

Nanoprobes are advantageous over small molecular probes in sensitivity for imaging applications but most luminescence molecules that used to construct nanoprobes often suffer from an aggregation-caused quenching(ACQ)effect.Ruthenium(?)tris(bipyridine)Ru(bpy)32+ and its derivatives are one type of novel phosphorescence probe with favorable photophysical properties.Notably,there was no obvious ACQ effect for Ru(bpy)32+ probes when they aggregate into nanoprobes.Nevertheless,nanoprobes always face the problems of reproducibility and difficulty of their fabrications,as well as targeting and cell membrane translocation when applied for cell imaging.Therefore,the ideal strategy is using a small molecular Ru(bpy)32+ probe for cell uptake but it can form nanoprobes inside cells,providing persistent phosphorescence imaging of the cells.But to the best of our knowledge,using a small molecular Ru(bpy)32+ probe to "smartly" self-assemble into nanoprobe with persistent phosphorescence inside cells has not been reported.Recently,Rao and co-workers developed a biocompatible,click condensation reaction between cysteine(Cys)and 2-cyanobenzothiazole(CBT),and used intracellular cues(protease,pH,or reduction)to initiate this reaction to self-assemble nanostructures in living cells.To date,this reaction has been widely explored and shown promising applications in protein labeling,molecular imaging(optical imaging,positron emission tomography imaging,and magnetic resonance imaging),intracellular hydrogelation,drug release,etc.Inspired by the above studies,we rationally designed a small molecular probe Cys(StBu)-Lys(Ru(bpy)32+)-CBT(1)which "smartly" self-assembled into nanoparticles 1-NPs inside cells with non-quenched,persistent phosphorescence.Employing this property,we successfully applied 1 for long time sensing of biothiol activity in living HepG2 cells and tumors.We envision that,by modifying the amino group with an enzyme substrate,our probe 1 could be further developed for sensing of intracellular enzyme activity with persistent,non-quenched phosphorescence.