Quantitative live-cell imaging of lipidated peptide transport through an epithelial cell layer.

Oral drug delivery increases patient compliance and is thus the preferred administration route for most drugs. However, for biologics the intestinal barrier greatly limits the absorption and reduces their bioavailability. One strategy employed to improve on this is chemical modification of the biologic through the addition of lipid side chains. While it has been established that lipidation of peptides can increase transport, a mechanistic understanding of this effect remains largely unexplored. To pursue this mechanistic understanding, end-point detection of biopharmaceuticals transported through a monolayer of fully polarized epithelial cells is typically used. However, these methods are time-consuming and tedious. Furthermore, most established methods cannot be combined easily with high-resolution live-cell fluorescence imaging that could provide a mechanistic insight into cellular uptake and transport. Here we address this challenge by developing an axial PSF deconvolution scheme to quantify the transport of peptides through a monolayer of Caco-2 cells using single-cell analysis with live-cell confocal fluorescence microscopy. We then measure the known cross-barrier transport of several compounds in our model and compare the results with results obtained in an established microfluidic model finding similar transport phenotypes. This verifies that already after two days the Caco-2 cells in our model form a tight monolayer and constitute a functional barrier model. We then apply this assay to investigate the effects of side chain lipidation of the model peptide drug salmon calcitonin (sCT) modified with 4‑carbon and 8‑carbon-long fatty acid chains. Furthermore, we compare that with experiments performed at lower temperature and using inhibitors for some endocytotic pathways to pinpoint how lipidation length modifies the main avenues for the transport. We thus show that increasing the length of the lipid chain increases the transport of the drug significantly but also makes endocytosis the primary transport mechanism in a short-term cell culture model.