Protein machineries defining pathways of nanocarrier exocytosis and transcytosis

The transport of nanocarriers through barriers like the gut in a living organism involves the transcytosis of these nanocarriers through the cell layer dividing two compartments. Understanding how this process works is not only essential to further developing strategies for a more effective nanocarrier transport system but also for providing fundamental insights into the barrier function as a means of protection against micro- and nanoplastics in the food chain. We therefore set out to investigate the different uptake mechanisms, intracellular trafficking and the routes for exocytosis for small polystyrene nanoparticles (PS-NPs ca. 100 nm) as mimicking nanocarriers in a Caco-2 cell model for gut-blood transition. We used label free, quantitative mass spectrometry (MS) for determining the proteome that adhered to transversed nanoparticles. From this rich proteomics dataset, as well as previous studies, we generated stabletransfected Caco-2 cell lines carrying the green fluorescent protein (GFP) coupled to proteins of interest for uptake, early, late and exocytotic endosomes. We detected the spatial and temporal overlap of such marked endosomes with the nanocarrier signal in confocal laser scanning and super-resolution microscopy. There was a clear distinction in the time course of nanoparticle trafficking between groups of proteins for endocytosis, intracellular storage and putatively transcytosis and we identified several key transcytotic markers like Rab3 and Copinel. Moreover, we postulate the necessity of a certain protein composition on endosomes for successful transcytosis of nanocarriers. Finally, we define the two-sided impasse of the lysosome as a dead end for nano-plastic and the limit of nanocarriers in the 100 nm range.(C) 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.