Dynamic in vitro intestinal barrier model coupled to chip-based liquid chromatography mass spectrometry for oral bioavailability studies

Abstract

In oral bioavailability studies, evaluation of the absorption and transport of drugs and food components across the intestinal barrier is crucial. Advances in the field of organ-on-a-chip technology have resulted in a dynamic gut-on-a-chip model that better mimics the in vivo microenvironment of the intestine. Despite a few recent integration attempts, ensuring a biologically relevant microenvironment while coupling with a fully online detection system still represents a major challenge. Herein, we designed an online technique to measure drug permeability and analyse unknown product formation across an intestinal epithelial layer of Caco-2 and HT29-MTX cells cultured on a flow-through Transwell system, while ensuring the quality and relevance of the biological model. Chip-based ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was coupled to the dynamic Transwell system via a series of switching valves, thus allowing alternating measurements of the apical and basolateral sides of the in vitro model. Two trap columns were integrated for online sample pre-treatment and compatibility enhancement. Temporal analysis of the intestinal permeability was successfully demonstrated using verapamil as a model drug and ergotamine epimers as a model for natural toxins present in foods. Evidence was obtained that our newly developed dynamic system provided reliable results versus classical static in vitro models, and moreover, for the first time, epimer-specific transport is shown for ergotamine. Finally, initial experiments with the drug granisetron suggest that metabolic activity can be studied as well, thus highlighting the versatility of the bio-integrated online analysis system developed.

A versatile, compartmentalised gut-on-a-chip system for pharmacological and toxicological analyses

“…In our previous work, a flow-through transwell barrier model was also coupled to MS detection, and sample dissolved in HBSS matrix was added to the apical chamber of the absorption module 23 . In this study, however, we have subjected our sample to in vitro digestion first, using the digestion compartment presented earlier 12 .…”

“…Dulbecco’s Modified Eagle Medium (DMEM) with 4.5 g/L glucose and L-glutamine with and without phenol red, bovine serum albumin (BSA) and heat-inactivated fetal bovine serum (FBS) were obtained from Gibco (Bleiswijk, the Netherlands). Rabbit polyclonal antibody ZO-1/TJP1 conjugated to Alexa fluor 594, Prolong Diamond Antifade Mountant, dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS) and non-essential amino acids (NEAA) were bought from Thermo Fisher Scientific (Landsmeer, the Netherlands) 23 . All chemicals for digestive juices, including enzymes, came from Sigma-Aldrich/Merck, except sodium dihydrogen phosphate monohydrate and hydrochloric acid (Acros, Geel, Belgium), and potassium chloride and sodium chloride (Duchefa, Haarlem, the Netherlands).…”

“…Barrier integrity was evaluated after cells had been cultured on a transwell membrane for 21 days and subsequently stained for tight junction protein, ZO-1, as described before 23 . Just before staining, the cells were washed with PBS and fixed with 4% paraformaldehyde (w/v) for 15 min, permeabilized with 0.25% Triton-X100 (v/v) and blocked with 1% BSA (w/v).…”

“…Alternatively, the flow-through transwell was connected to Compartment 3, which consisted of a series of three switching valves connected to a microfluidic chip-based UPLC-QTOF-MS (Fig. 1 c) as described by Santbergen et al 23 In this latter option, apical and basolateral effluents from the flow-through transwell were alternately loaded in 5 µL stainless steel sample loops mounted on the first and second switching valve (Fig. 1 c).…”

“…In the case of verapamil analysis, the C8 nanotrap column was eluted towards a microfluidic chip-based iKey BEH C18 analytical column using the following gradient. The 3 µL/min microflow gradient was based on a published method 23 and consisted of mobile phase A (water with 1% acetonitrile) and mobile phase B (acetonitrile with 1% water), both containing 0.1% formic acid. The gradient started at 10% B and, after 4 min, was linearly increased to 100% B in 4 min.…”