Protein corona fingerprinting of exocytosed nanoparticles reveals time-dependence of exocytosis pathways.

Abstract

In the field of nano-bio interactions, the cellular uptake of nanocarriers and the modulating factors, e.g. the presence of a protein shell - the protein corona - adsorbed on the nanocarrier surface are well studied. However, little is known about the detailed mechanisms by which nanocarriers leave a cell - the exocytosis processes of nanocarriers from cells - and their driving determinants. Here, we analyzed the protein components of magnetic dextran-coated iron oxide particles exocytosed from a human tumor cell model over time using mass spectrometry-based proteomics and bioinformatic annotation analysis to map the dynamics of the nanocarrier exocytosis mechanism. We demonstrated that the protein corona desorbed from exocytosed particles can serve as a molecular fingerprint of the exocytosis pathways involved. We correlated the proteomic findings mainly using ultrastructural imaging, but also with other conventional methods such as inductively coupled plasma optical emission spectrometry, flow cytometry, and confocal microscopy. Our data showed that the presence of a pre-coated human plasma protein corona did not drastically affect the extent and route of exocytosis. Most importantly, we demonstrated the time-dependency of a non-conventional exocytosis mechanism for the iron oxide particles investigated with a major contribution of lysosomal exocytosis accompanied by secretion of extracellular vesicles and extracellular vesicle proteins. STATEMENT OF SIGNIFICANCE: Nanocarriers are not only internalized by cells but also undergo exocytosis. We show that their intracellular fate can be traced via protein signatures on their surface post-exocytosis. Analyzing this protein corona is challenging due to limited nanoparticle recovery, but label-free quantitative liquid chromatography-mass spectrometry (LC-MS) enabled identification of pathway-specific protein fingerprints. Clustering these fingerprints revealed distinct intracellular trafficking routes and exocytosis mechanisms, highlighting the complexity of both processes. Transmission electron microscopy (TEM) validated nanocarrier transit through cellular compartments. Notably, we observed time-dependent involvement of an unconventional exocytosis mechanism, predominantly lysosomal-like exocytosis. This integrative approach deciphers nanocarrier behavior, linking surface biomolecular profiles to intracellular dynamics.