Nanoparticle internalisation is crucial for the precise delivery of drug/genes to its intracellular targets. Conventional quantification strategies can provide the overall profiling of nanoparticle biodistribution, but fail to unambiguously differentiate the intracellularly bioavailable particles from those in tumour intravascular and extracellular microenvironment. Herein, we develop a binary ratiometric nanoreporter (BiRN) that can specifically convert subtle pH variations involved in the endocytic events into digitised signal output, enabling the accurately quantifying of cellular internalisation without introducing extracellular contributions. Using BiRN technology, we find only 10.7–28.2% of accumulated nanoparticles are internalised into intracellular compartments with high heterogeneity within and between different tumour types. We demonstrate the therapeutic responses of nanomedicines are successfully predicted based on intracellular nanoparticle exposure rather than the overall accumulation in tumour mass. This nonlinear optical nanotechnology offers a valuable imaging tool to evaluate the tumour targeting of new nanomedicines and stratify patients for personalised cancer therapy.

Precise monitoring of the subtle pH fluctuation during biological events remains a big challenge. Previously, we reported an ultra-pH-sensitive (UPS) nanoprobe library with a sharp pH response using copolymerization of two tertiary amine-containing monomers with distinct pK a . Currently, we have generalized the UPS nanoparticle library with tunable pH transitions (pH t ) by copolymerization of a tertiary amine-containing monomer with a series of non-ionizable monomers. The pH t of nanoparticles is fine-tuned by the non-ionizable monomers with different hydrophobicity. Each nonionizable monomer presents a constant contribution to pH tunability regardless of tertiary amine-containing monomers. Based on this strategy, we produced two libraries of nanoprobes with continuous pH t covering the entire physiological pH range (5.0-7.4) for fluorescent imaging of endosome maturation and cancers. This generalized strategy provides a powerful toolkit for biological studies and cancer theranostics.

Precise monitoring of the subtle pH fluctuation during biological events remains a big challenge. Previously, we reported an ultra‐pH‐sensitive (UPS) nanoprobe library with a sharp pH response using co‐polymerization of two tertiary amine‐containing monomers with distinct pKa. Currently, we have generalized the UPS nanoparticle library with tunable pH transitions (pHt) by copolymerization of a tertiary amine‐containing monomer with a series of non‐ionizable monomers. The pHt of nanoparticles is fine‐tuned by the non‐ionizable monomers with different hydrophobicity. Each non‐ionizable monomer presents a constant contribution to pH tunability regardless of tertiary amine‐containing monomers. Based on this strategy, we produced two libraries of nanoprobes with continuous pHt covering the entire physiological pH range (5.0–7.4) for fluorescent imaging of endosome maturation and cancers. This generalized strategy provides a powerful toolkit for biological studies and cancer theranostics.