title: 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 0.6-900kDa 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 (55kDa) and IgG (160kDa) 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.
authors.affiliation: Morehouse School of Medicine, Atlanta, GA, United States; Division of Pulmonary Allergy, Critical Care and Sleep Medicine, Department of Medicine, Emory University School of Medicine, United States.
authors.forename: Tarianna
authors.initials: T
authors.lastname: Stewart
authors.affiliation: Division of Pulmonary Allergy, Critical Care and Sleep Medicine, Department of Medicine, Emory University School of Medicine, United States.
authors.forename: William T
authors.initials: WT
authors.lastname: Koval
authors.affiliation: Division of Pulmonary Allergy, Critical Care and Sleep Medicine, Department of Medicine, Emory University School of Medicine, United States.
authors.forename: Samuel A
authors.initials: SA
authors.lastname: Molina
authors.affiliation: Kimberly-Clark Corporation, Atlanta, GA, United States.
authors.forename: Suzanne M
authors.initials: SM
authors.lastname: Bock
authors.affiliation: Morehouse School of Medicine, Atlanta, GA, United States.
authors.forename: James W
authors.initials: JW
authors.lastname: Lillard
authors.affiliation: Kimberly-Clark Corporation, Atlanta, GA, United States.
authors.forename: Russell F
authors.initials: RF
authors.lastname: Ross
authors.affiliation: Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA, United States.
authors.forename: Tejal A
authors.initials: TA
authors.lastname: Desai
authors.affiliation: Division of Pulmonary Allergy, Critical Care and Sleep Medicine, Department of Medicine, Emory University School of Medicine, United States; Department of Cell Biology, Emory University School of Medicine, Atlanta, GA, United States. Electronic address: mhkoval@emory.edu.
authors.forename: Michael
authors.initials: M
authors.lastname: Koval
mesh_terms: D018938:Caco-2 Cells; D045744:Cell Line, Tumor; D016503:Drug Delivery Systems; D004847:Epithelial Cells / Q000187:drug effects*; D004848:Epithelium / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D049329:Nanostructures / Q000737:chemistry*; D013499:Surface Properties; D057900:Transcytosis / Q000187:drug effects*
keywords: Calcium switch; Drug delivery; Epithelial permeability; FcRn; Nanotopography; Tight junctions; Transcytosis