Precise visualization of nanoparticles (NPs) at the subcellular level is crucial for targeted drug delivery. However, NPs functionalized with permanently fluorescent probes are challenging to detect when only a small fraction reaches the target compartment, as weak fluorescence signals are obscured by intense fluorescence from NPs accumulated in off-target subcellular compartments. Therefore, the aim of this work was to establish fluorogenic NPs that activate their fluorescence only at the intracellular target upon the inverse electron demand Diels-Alder (iEDDA) click reaction. The fluorogenic moiety on the NP surface was a coumarin-102-tetrazine (C102-Tet), while trans-cyclooctene (TCO) served as the second reaction partner immobilized on microtubules using anchors of different geometries and spatial accessibilities. We generated polymer NPs composed of poly(lactide-co-glycolide) (PLGA) and poly(ethylene glycol)-poly(lactic acid) (PEG-PLA) by systematically varying the PEG chain length (2k vs 5k) and C102-Tet surface density. Direct stochastic optical reconstruction microscopy (dSTORM) resolved individual NPs of ∼80 nm and confirmed that C102-Tet presented on the NP surface remains reactive. Quantitatively, 15-45% of the C102-Tet groups participated in the iEDDA reaction, depending on PEG chain length and surface density. To overcome insufficient endosomal escape, which is a still-unsolved challenge for nano-sized drug delivery systems, NPs were directly delivered into the cytosol to prove the fluorogenic NP concept that becomes activated only upon bio-orthogonal reaction at the intracellular target. Confocal microscopy and super-resolution nonlinear structured illumination microscopy imaging revealed NP fluorescence aligned along microtubules and supported target-specific iEDDA activation on the surface of NPs. This strategy enables high-contrast tracking of nanocarriers at defined intracellular targets.
