Abstract
Absorption and secretion of peptide and protein cargoes across single-cell thick mucosal and endothelial barriers occurs by active endocytic and vesicular trafficking that connects one side of the epithelial or endothelial cell (the lumen) with the other (the serosa or blood). Assays that assess this pathway must robustly control for non-specific and passive solute flux through weak or damaged intercellular junctions that seal the epithelial or endothelial cells together. Here we describe an in vitro cell culture Transwell assay for transcytosis of therapeutic peptides linked covalently to various species of the glycosphingolipid GM1. We recently used this assay to develop technology that harnesses endogenous mechanism of lipid sorting across epithelial cell barriers to enable oral delivery of peptide and protein therapeutics.
Keywords: Transcytosis, Endocytic sorting, Glycosphingolipids, Epithelial barriers, Drug delivery
Background
Transport of large molecules across the single-cell thick epithelial barriers lining mucosal surfaces and tight endothelial barriers lining vessels serving the heart muscle and brain occurs by an endocytic process that connects one side of these polarized cells with the other. This process is termed transcytosis (Garcia-Castillo et al., 2017). The absorption and secretion of immunoglobulins by receptor-mediated endocytosis and vesicular transport across the mucosa of the alimentary and respiratory tracts most famously typifies this process. Interest in transcytosis is also stimulated by the potential to harness this pathway for the delivery of therapeutic peptides and proteins across tight epithelial and endothelial barriers (Thuenauer et al., 2017).Transcytosis is an active (ATP-driven) process. In some cases, transport of large molecules across tight epithelial and endothelial barriers can occur by passive diffusion around the cell through intercellular tight junctions (Fung et al., 2018). But aside from this small level of paracellular leakiness, physiologic meaningful paracellular transport of large molecules around cells occurs only when intercellular tight junctions are dismantled–usually under pathologic conditions. Non-specific and low levels of transcellular transport for large solutes may also occur by fluid-phase endocytosis and miss-sorting of cargo into transcytotic vesicles rather than into the late endosome/lysosome pathway, which receives the bulk of cargo internalized by non-receptor mediated mechanisms. Thus, any assay for transcytosis across healthy epithelial or endothelial barriers in vitro or in vivo needs to control for these confounding non-specific pathways. We have recently discovered that the endogenous sorting of glycosphingolipids across epithelial barriers can be harnessed for oral delivery of therapeutic peptides in vivo (Garcia-Castillo et al., 2018). Here we describe the in vitro assay using cultures polarized epithelial cells grown on transwell filters that we used to develop the technology that co-opts these endogenous mechanisms of lipid sorting for this purpose. The test compounds used to harness glycolipid sorting are modified to contain a biotin residue to allow for biochemical capture and a fluorophore for detection. Transwell inserts are composed of an inner chamber made with a permeable polycarbonate membrane support where the cells are seeded (Figure 1). After 3-7 days the epithelial cells assemble into a single cell thick monolayer with sealed tight junctions among cells. As such, the inner chamber of the transwell is exposed to the apical membrane of the epithelial monolayer and models the lumenal surface and can hold 200 µl of solution. The outer chamber–the chamber below the permeable support–is exposed to the basolateral surface of the monolayer and models the serosal surface of the epithelial barrier. This chamber holds 1 ml of basolateral solution. The assembly of the monolayer into a “tight” epithelial barrier with sealed functional tight junctions is routinely measured by passive resistance to small ion transport (TER–Transepithelial Resistance) measured by standard direct current electrophysiology or using the alternate current based machine EVOM. Test compounds are added to the apical chamber, and the assay is allowed to proceed to allow for transport across the barrier, where the basolateral chamber is then sampled for the transcytosed analyte using streptavidin-coated beads (Figure 2). Figure 1. Schematic representation of MDCK-II transwell inserts and transcytosis assayFigure 2. Capture and read of biotinylated peptide–fusions from basolateral media (Adapted from Figure 1A in Garcia-Castillo et al., 2018. Creative Commons Attribution License)Mid-picomolar sensitivity can be achieved with this assay. Our basic approach can be used to measure transport by other molecules that enter the transcytotic pathway–such as for IgG that binds to the Fcγ-receptor FcRn that traffics in both directions across polarized epithelial monolayers (Nelms et al., 2017). The strength of our assay is the ability to directly, quantitatively, and sensitively measure the amount of compound transcytosed. Non-specific paracellular leak between cells versus real transport through cells is controlled in several ways, such as by using an analyte that cannot engage the transcytotic pathway (in our case the reporter peptide lacking fusion to the glycolipid carrier, or by fusion to a glycolipid with ceramide structure that cannot enter the transcytotic pathway), or by inhibiting endocytosis or transcytosis via a 4 °C temperature block, by chemical inhibition of endocytosis using Dyngo, and by siRNA knockdown of genes responsible for transcytosis as described in Garcia-Castillo et al. (2018).
Materials and Reagents
Notes:
Equipment
Software
Procedure
Data analysis
GraphPad Prism software is used to calculate the concentration of lipid-peptide fusion. As detailed above, a standard curve for each test compound is used to relate fluorescence intensity to a known concentration of peptide or lipid-peptide fusion. Absorbance at 495 nm is initially used to calculate stock concentrations. For detailed instructions on graphing and interpolating standard curves, the reader is referred to the GraphPad software website. To calculate the apparent permeability coefficient (PAPP, cm/sec) a standard curve ranging from 0 nM to 200 nM is used to interpolate concentration of peptide or lipid-peptide solution remaining in the apical chamber after a 3 h continuous incubation. The apparent permeability coefficient (PAPP, cm/sec) is calculated across cell monolayers grown in transwells based on the appearance rate of lipid-peptide fusions in the basolateral compartment over time: Papp (cm/sec) = [VD/(A x MD)] x (DMR/Dt) Papp (cm/sec) = [cm3/(cm2 x mol)] x (mol/sec) VD = apical (donor) volume (cm3) (e.g., 0.2 ml = 0.2 cm3) MD = apical (donor) amount (mol) A = membrane surface area (cm2) of apical (donor) chamber (i.e., transwell surface area = 0.33 cm2 in this protocol) DMR/Dt = the amount of compound (mol) transferred to the basolateral (receiver) compartment over time (sec).
Notes
Recipes
Acknowledgments
This project was supported by an NIH F-32 DK111072-01 to M.D.G-C; DK084424, DK048106, DK104868, and an unrestricted innovator grant from Novo Nordisk to W.I.L; DK104868 to D.C. We acknowledge the Harvard Digestive Disease Center DK034854. We are grateful to all members of the Lencer and Chinnapen laboratories for their helpful discussions.
Competing interests
The authors declare that there are no conflicts of interest or competing interests.
References
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