Molecular Mechanisms of Intracellular Delivery of Nanoparticles Monitored by an Enzyme-Induced Proximity Labeling.

Achieving increasingly finely targeted drug delivery to organs, tissues, cells, and even to intracellular biomacromolecules is one of the core goals of nanomedicines. As the delivery destination is refined to cellular and subcellular targets, it is essential to explore the delivery of nanomedicines at the molecular level. However, due to the lack of technical methods, the molecular mechanism of the intracellular delivery of nanomedicines remains unclear to date. Here, we develop an enzyme-induced proximity labeling technology in nanoparticles (nano-EPL) for the real-time monitoring of proteins that interact with intracellular nanomedicines. Poly(lactic-co-glycolic acid) nanoparticles coupled with horseradish peroxidase (HRP) were fabricated as a model (HRP(+)-PNPs) to evaluate the molecular mechanism of nano delivery in macrophages. By adding the labeling probe biotin-phenol and the catalytic substrate H2O2 at different time points in cellular delivery, nano-EPL technology was validated for the real-time in situ labeling of proteins interacting with nanoparticles. Nano-EPL achieves the dynamic molecular profiling of 740 proteins to map the intracellular delivery of HRP (+)-PNPs in macrophages over time. Based on dynamic clustering analysis of these proteins, we further discovered that different organelles, including endosomes, lysosomes, the endoplasmic reticulum, and the Golgi apparatus, are involved in delivery with distinct participation timelines. More importantly, the engagement of these organelles differentially affects the drug delivery efficiency, reflecting the spatial-temporal heterogeneity of nano delivery in cells. In summary, these findings highlight a significant methodological advance toward understanding the molecular mechanisms involved in the intracellular delivery of nanomedicines.

Fast and Dynamic Mapping of the Protein Corona on Nanoparticle Surfaces by Photocatalytic Proximity Labeling.

Protein corona broadly affects the delivery of nanomedicines in vivo. Although it has been widely studied by multiple strategies like centrifugal sedimentation, the rapidly forming mechanism and the dynamic structure of the protein corona at the seconds level remains challenging. Here, a photocatalytic proximity labeling technology in nanoparticles (nano-PPL) is developed. By fabricating a "core-shell" nanoparticle co-loaded with chlorin e6 catalyst and biotin-phenol probe, nano-PPL technology is validated for the rapid and precise labeling of corona proteins in situ. Nano-PPL significantly improves the temporal resolution of nano-protein interactions to 5 s duration compared with the classical centrifugation method (>30 s duration). Furthermore, nano-PPL achieves the fast and dynamic mapping of the protein corona on anionic and cationic nanoparticles, respectively. Finally, nano-PPL is deployed to verify the effect of the rapidly formed protein corona on the initial interaction of nanoparticles with cells. These findings highlight a significant methodological advance toward nano-protein interactions in the delivery of nanomedicines in vivo.

Nanoparticulates reduce tumor cell migration through affinity interactions with extracellular migrasomes and retraction fibers.

Nano-tumor interactions are fundamental for cancer nanotherapy, and the cross-talk of nanomedicines with the extracellular matrix (ECM) is increasingly considered essential. Here, we specifically investigate the nano-ECM interactivity using drug-free nanoparticulates (NPs) and highly metastatic cancer cells as models. We discover with surprise that NPs closely bind to specific types of ECM components, namely, retraction fibers (RFs) and migrasomes, which are located at the rear of tumor cells during their migration. This interaction is observed to alter cell morphology, limit cell motion range and change cell adhesion. Importantly, NPs are demonstrated to inhibit tumor cell removal in vitro, and their anti-metastasis potential is preliminarily confirmed in vivo. Mechanically, the NPs are found to coat and form a rigid shell on the surface of migrasomes and retraction fibers via interaction with lipid raft/caveolae substructures. In this way, NPs block the recognition, endocytosis and elimination of migrasomes by their surrounding tumor cells. Thereby, NPs interfere with the cell-ECM interaction and reduce the promotion effect of migrasomes on cell movement. Additionally, NPs trigger alteration of the expression of proteins related to cell-cell adhesion and cytoskeleton organization, which also restricts cell migration. In summary, all the findings here provide a potential target for anti-tumor metastasis nanomedicines.