br Acknowledgments I thank Takeshi Sakaba for
Acknowledgments I thank Takeshi Sakaba for critical reading of the manuscript and helpful comments. This work was supported by JSPS/MEXT KAKENHI Grant Numbers 17K19466, 17H03548.
Introduction The oral route of drug application is the main route in pharmacology since it is easy to handle and overall more comfortable for the patient compared to an intravenous application . But not all drugs survive the harsh and intentionally digestive conditions in the stomach and gut. Especially proteins or peptides are prone to destruction by digestion. Furthermore, many macromolecules are poorly absorbed in the intestine due to their chemical properties . Here, nanocarriers offer the unique feature to protect the cargo. The feasibility of such an approach has been shown by the encapsulation of insulin , . The clear disadvantage of the oral route is its low efficiency of transcytosis of nanocarriers in the gut. This can only be overcome by a comprehensive knowledge of the transport processes involved of which transcytosis is an essential step. From a different perspective, the restrictive barrier function of the gastrointestinal (GI) tract is an essential for protection against all different kind of food ingredients. In the last years, the scientific and public awareness of environmental pollution of micro – (0.1–5000 µm) and nanoplastics (1–100 nm) in food and drinking water has drawn increased attention. However there is a lack of toxicity and toxicokinetic data in humans , especially for nanoplastics. Studies on local effects on the GI tract e.g. with polymeric nanoparticles as a mimic for nanoplastic  and suitable cellular models could provide fundamental knowledge. Understanding the routes through the cell barrier is the key for developing nanocarrier systems and provide data for the risk assessment of nanoplastics on the other side, depending on studied nanomaterial (size, charge, surface chemistry, protein corona). Either a route between the cells, namely paracellular transport , or directly through the gut epithelial RO4987655 sale needs to be taken. The last process is termed transcytosis. While ions or small molecules can pass the cell by entering and leaving the cell plasma via specific transmembrane channels, nanocarriers use transcytosis involving membrane wrapped endosomal structures in a partially energy-dependent process . Transcytosis can be perceived as a combination of endocytosis and exocytosis. This process involves several up to now not accurate characterized intracellular endosomal structures. Within the last years, many studies focused on the influence of different nanoparticle properties on transcytosis efficiencies. While size , charge  and functionalization , ,  of the nanomaterial have a major impact on the rate of transcytosis as well as exocytosis , , a well-founded knowledge behind the exact mechanism of a nanocarrier entering and passing an epithelial cell layer is up to now not available. The majority of studies so far focused on endocytosis pathways of nanoparticles in intestinal cells, discussing clathrin , , , - or caveolae/lipid raft , , -mediated endocytosis as well as macropinocytosis , , depending on the material of the nanocarrier. Our aim in this study was to establish a method for the investigation of a nanocarrier’s way through the cell. We therefore set out to first determine proteins associated with transcytosed nanocarriers in Caco-2 cells, which are widely used as the standard for gut-blood transition experiments . We synthesized carboxyl-functionalized polystyrene nanoparticles (PS-COOH-NP) with a size of approximately 100 nm via direct miniemulsion polymerization. By label-free, quantitative mass spectrometry we determined proteins attached on these transcytosed nanocarriers during their passage through the cell as well as basal secreted proteins. From the list of proteins as well as from previous studies  we selected three different kind of proteins: Proteins so far assigned with the endolysosomal pathway, proteins with a putative role in exocytosis, and proteins hitherto not linked to transcytosis but prominently found in our proteomics data. So we dissected the transversing route of the nanocarrier through the cell. By evaluating the co-localization of fluorescently labeled endosomal compartments and nanocarrier we were able to determine the dynamic trafficking from early endosomes to late endosomes and from there to the putatively exocytosis related endosomal compartment. Importantly, we demonstrate how the nanocarrier is mainly trafficked to the intracellular waste compartment, termed the lysosome. With the lysosome being a dead end inside the cell, exocytosis will unlikely occur anymore. While up to now we cannot provide a solution to this, our approach pinpoints this problem. Our methodology also demonstrates that by observing different patterns of the endosomal intracellular dynamics hitherto unclassified marker proteins can be found. By expanding the methodology of selecting intracellular compartments and following their fate we will ultimately be able to understand and manipulate the intracellular sorting process.