Elucidating the dynamic interactions between nanocarriers and cellular machinery is critical for advancing targeted nanomedicine. However, optical microscopy imaging techniques can only provide a generalized view of nanomedicine localization, while proteomics approaches require cell lysis that disrupts native protein coronas and obscures real-time intracellular trafficking mechanisms. Although proximity labeling enables in situ investigation of intracellular protein-protein interactions, it relies on genetically engineered enzyme fusion, limiting its applicability across diverse systems.
To address this challenge, a research team led by Prof. YUAN LIU and Prof. JI JING from Hangzhou Institute of Medicine (HIM) of the Chinese Academy of Sciences, and Prof. YUNLU DAI from the University of Macau developed a genetic-engineering-free strategy called nanozyme proximity labeling (NPL) to map the in situ interactomes and trafficking pathways of nanoparticles in live cells. The research was published in Proceedings of the National Academy of Sciences (PNAS).
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 APEX. Isolating labeled proteins and analyzing them by mass spectrometry enabled the identification of proteins that interact with the nanozyme in the native cellular environment.
By comparing the in situ interactomes of mitochondria-targeted versus non-targeted nanoparticles, the researchers found that mitochondria-targeted nanoparticles exhibited a 1.5‑fold enrichment of mitochondrial proteins and engaged intracellular trafficking mediators that facilitated their anchorage to mitochondria, whereas non-targeted nanoparticles were predominantly routed through lysosomal degradation pathways. This work provides a high-resolution, in situ snapshot of how surface modifications dictate subcellular fate.
The NPL platform requires no genetic modification and can be broadly applied to dissect nanomedicine-bio interfaces. By swapping different peroxidase-like nanoparticles or modifying distinct targeting ligands, this strategy enables the study of diverse intracellular trafficking pathways and interaction networks, providing a powerful tool for the rational design and precise optimization of nanomedicines.
