The epithelial cells of the intestine serve as a key barrier governing the uptake of orally administered therapeutic compounds, thus having a profound impact on the bioavailability of these drugs. At present, however, there is a lack of any comprehensive research regarding cellular responses relating to nanoparticle transport or feedback mechanisms activated in the context of transcytosis across the epithelial cell monolayer. To explore these mechanisms, we therefore used Madin−Darby canine kidney (MDCK) cells and zebrafish as models for evaluating the transport of poly(ethylene glycol)-b-poly (trimethylene carbonate) (PEG-PTMC) nanoparticles, studying their endocytic uptake, trafficking within cells, exocytosis, and transcytosis. A label-free approach to proteomic quantification was used in order to gain comprehensive insights into cellular responses to continuous endocytosis via analysis of cells following cellular uptake. This approach was further used to compare endocytosis and transcytosis responses. Given its high fluorescence and poor solubility, coumarin 6 (C6) was selected in this study as a model hydrophobic drug which was used to generate C6-labeled PEG-PTMC nanoparticles. These nanoparticles were readily trafficked in vitro and in vivo, thus offering novel insights into nanoparticles trafficking processes. These obtained results thus provide new information regarding efficient nanodelivery across epithelial barriers.