Calibrated flux measurements reveal a nanostructure-stimulated transcytotic pathway

Abstract

Transport of therapeutic agents across epithelial barriers is an important element in drug delivery. Transepithelial flux is widely used as a measure of transit across an epithelium, however it is most typically employed as a relative as opposed to absolute measure of molecular movement. Here, we have used the calcium switch approach to measure the maximum rate of paracellular flux through unencumbered intercellular junctions as a method to calibrate the flux rates for a series of tracers ranging in 0.6 to 900 kDa in size across barriers composed of human colon epithelial (Caco-2) cells. We then examined the effects of nanostructured films (NSFs) on transepithelial transport. Two different NSF patterns were used, Defined Nanostructure (DN) 2 imprinted on polypropylene (PP) and DN3 imprinted on polyether ether ketone (PEEK). NSFs made direct contact with cells and decreased their barrier function, as measured by transepithelial resistance (TER), however cell viability was not affected. When NSF-induced transepithelial transport of Fab fragment (55 kDa) and IgG (160 kDa) was measured, it was unexpectedly found to be significantly greater than the maximum paracellular rate as predicted using cells cultured in low calcium. These data suggested that NSFs stimulate an active transport pathway, most likely transcytosis, in addition to increasing paracellular flux. Transport of IgG via transcytosis was confirmed by immunofluorescence confocal microscopy, since NSFs induced a significant level of IgG endocytosis by Caco-2 cells. Thus, NSF-induced IgG flux was attributable to both transcytosis and the paracellular route. These data provide the first demonstration that transcytosis can be stimulated by NSFs and that this was concurrent with increased paracellular permeability. Moreover, NSFs with distinct architecture paired with specific substrates have the potential to provide an effective means to regulate transepithelial transport in order to optimize drug delivery.

Nanotopography enhances dynamic remodeling of tight junction proteins through cytosolic complexes

“…1C-E ). These data are consistent with our previous studies, which demonstrate increased paracellular permeability to macromolecules when epithelial cells are in contact with nanotopographic structures ( 12-14 ), further indicating the regulation of tight junctions through nanostructure contact. We then immunostained ZO-1 in differentially treated cells (fig.…”

“…Tight junctions are highly dynamic, even in unstimulated cells, and are acutely regulated by multiple extracellular biological stimuli including hormones and cytokines ( 2, 4 ). We previously found in vitro and in vivo that cell contact with polymeric films with defined nanotopographic features enhances transepithelial permeability to macromolecules in the size range of (60 kDa to 150 kDa) ( 12-14 ). Here, for the first time, we used state-of-the-art TIRF microscopy to visualize paracellular flux of FITC-labeled IgG with submicron resolution at the basal aspect of live epithelial monolayers stimulated with a nanostructured biophysical cue on the apical surface.…”

“…Nanostructured films were made, as previously described ( 12-14 ), by nanoimprint lithography in which polypropylene was heated above its glass transition temperature and pressed into a silicon mold. Molds were fabricated using electron beam lithography followed by anisotropic reactive ion etching to generate precise submicron structures.…”

“…Synthetic materials fabricated with specific geometries and surface topographic features at the micro and nanoscale have the capacity to influence epithelial cell behavior ( 10, 11 ). We previously found that polymeric films with defined nanostructure, when placed in contact with the apical aspect of an epithelial monolayer, led to enhanced transepithelial permeability of macromolecules ranging in size from ∼60 kDa to ∼150 kDa, including bovine serum albumin and IgG ( 12-14 ). Immunolabeling of ZO-1 showed characteristic changes in tight junction morphology that were associated with increased permeability, further indicating nanotopography-induced regulation of tight junctions ( 12 ).…”

Sphingomyelinase decreases transepithelial anion secretion in airway epithelial cells in part by inhibiting CFTR‐mediated apical conductance

“…Paracellular flux was measured using calcein (Schlingmann et al, 2016; Stewart et al, 2017). Krebs Ringers HEPES (KRH) buffer was 150 mM NaCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM glucose, 10 mM HEPES, and pH 7.4.…”