Elucidating dynamic interactions between nanocarriers and cellular machinery is critical for advancing targeted nanomedicine. Optical microscopy imaging techniques only provide a generalized view of nanomedicine localization. Proteomics approaches require cell lysis which disrupt native protein coronas during isolation, obscuring real-time intracellular trafficking mechanisms.

Proximity labeling enables in situ investigation of intracellular protein-protein interactions, but it relies on genetically engineered enzyme fusion, which limits its applicability across diverse systems.

In a study published in PNAS, a research team led by Prof. LIU Yuan and Prof. JING Ji from Hangzhou Institute of Medicine (HIM) of the Chinese Academy of Sciences, and Prof. DAI Yunlu from the University of Macau, developed a genetic-engineering-free strategy, nanozyme proximity labeling (NPL), to map the in situ interactomes and trafficking pathways of nanoparticles in live cells.

The researchers leveraged iron oxide (Fe₃O₄) nanoparticles with peroxidase-like activity to covalently label proximal proteins in situ within just one minute upon hydrogen peroxide activation, similar to Ascorbate Peroxidase based proximity labeling. By isolating labeled proteins and analyzing them through mass spectrometry, they identified proteins that interact with the nanozyme in the native cellular environment.

Moreover, the researchers compared in situ interactomes of mitochondria-targeted versus non-targeted nanoparticles. Mitochondria-targeted nanoparticles exhibited a 1.5‑fold enrichment of mitochondrial proteins and engaged intracellular trafficking mediators which facilitated their anchorage to mitochondria. Non-targeted nanoparticles were mainly routed through lysosomal degradation pathways.

This work provides a high-resolution, in situ snapshot of how surface modifications dictate subcellular fate. The NPL strategy requires no genetic modification and can be applied to dissect nanomedicine-bio interfaces. It enables the study of diverse intracellular trafficking pathways and interaction networks, providing a powerful tool for the rational design and precise optimization of nanomedicines.