Small intestinal epithelial cell models designed for utilizing advanced microscopic platforms to investigate transport of biopharmaceuticals

The oral route displays the most convenient way of administrating a drug due to high patient compliance and non-invasiveness. However, this route is characterized by substantial challenges created by the highly degradative nature of the gastrointestinal tract (GIT) and the first-pass metabolism in the liver, leading to poor bioavailabilities of orally administered drugs. Especially, peptide and protein drugs, which are associated with low off-target effects based on their high targeting specificity, are hindered for successful transport across the GIT into the systemic circulation as a result of their size and metabolic instability. To improve the transport across the intestine several attempts have been made by modifying the peptide itself, formulating it into a carrier system, or by the co-administration with permeation enhancer. However just five orally administered peptide drugs are currently approved by the Food and Drug Administration (FDA). The unsuccessful clinical translation of peptide drugs is associated with insufficient knowledge of the underlying mechanism of transport across the intestinal epithelium, which is linked to a strong propensity to rely on end-point in vitro screening assays. The implementation of live cell imaging techniques could provide novel insights into the drug-barrier interaction and thus improve the screening of drug candidates in the early stage of preclinical drug development. Nevertheless, common in vitro models hinder the use of live cell imaging due to their design. In order to, investigate the absorption mechanism of peptide drugs in more detail, novel in vitro platforms resembling the human epithelial cell barrier are of great need.
In this PhD thesis, three different in vitro models reflecting the physiological epithelial barrier of the small intestine were developed and utilized as imaging-compatible platforms for in-depth investigations of peptide drug absorption.
For the first in vitro model, an imaging-compatible microfluidic chip system was used for the cultivation of Caco-2 and HT29-MTX E12 cells, mimicking enterocytes and goblet cells, resulting in the cultivation of 3D tubular epithelial monolayers. The generated epithelial tubules were characterized by Spinning disk microscopy (SD) and transmission electron microscopy (TEM), demonstrating fully differentiated and polarized monolayers. Next, the imaging compatibility of the chip system was utilized for establishing a quantitative fluorescent-based real-time transport assay for the investigation of biologics. The implementation allowed for high-temporal resolution tracking of leading peptides within the field of oral drug delivery TAT, Insulin and Semaglutide, with the ability for simultaneous monitoring of the barrier properties.
A limitation of in vitro assays relying on cancer cell lines like Caco-2 is that they poorly mimic the diverse cell types and state of the intestinal epithelium in vivo. Thus, to better resemble the in vivo like scenario, a novel intestinal organoids-based in vitro assay was developed, characterized and employed. Intestinal organoids hold great potential as a screening platform due to their multicellular landscape reflecting the native intestinal physiology, but the inaccessibility of the absorptive site impedes their use in drug absorption studies. In this PhD thesis, apical-out organoids were developed using a suspension culture method and thus enabled access to the apical site. The apical-out organoids were characterized for phenotypical markers using SD and TEM and finally deployed for the investigation of the uptake and transport of TAT and Insulin by live cell imaging. To enhance the throughput of structurally defined apical-out organoids a biocompatible micropillar device was developed for the entrapment of individual organoids during the cultivation process. The device further enables the use as a multipoint image-based drug screening platform.
Since the intestinal barrier is not just an unattached cell layer but rather describes a dynamic interface with other tissues, an in vitro system resembling the tissue-tissue interface of the epithelium and lacteal in the small intestine was established. Therefore, Caco-2 cells and lymphatic endothelial cells were cultivated in the same microfluidic chip system, forming 3D tubular structures, which are connected by an extracellular matrix. The in vitro model was used to study the oral lymphatic drug transport via chylomicrons.
In summary, all established in vitro barrier models are encompassing the potential for bridging the translational gap between preclinical and clinical studies of peptide drug candidates, fostering the collection of in-depth mechanistic insights in intestinal drug uptake and transport.