Abstract by Danai Anastasia Panou
Oral delivery of peptide drugs can be considered a life-changing solution for patients for whom treatment is dependent on injections. Despite the current progress in peptide and formulation designs to overcome some of the main challenges in the environment of the gastrointestinal tract, inefficient absorption through the intestinal epithelium remains a major obstacle. A promising strategy to tackle the latter is to apply excipients that can modulate the tight junctions (TJs); the protein network responsible for regulating the paracellular pathway in the intestinal epithelium. Therefore, the present thesis explored the modulation of TJ and cytoskeleton dynamics as a strategy for oral peptide delivery. The overall aims of this project were (i) to study the mechanisms by which the membrane-interacting peptide excipient, penetramax, an analog of the cell-penetrating peptide, penetratin, exerts its transepithelial enhancing properties and elucidate its effect on TJ and cytoskeleton dynamics in vitro, and, (ii) to compare penetramax and ethylene glycol-bis(2-aminoethylether)-N,N,Nʹ,Nʹ-tetraacetic acid (EGTA) head-to-head, by elucidating differences in their permeation enhancement time-profile and correlate this with their effect on TJ and cytoskeleton dynamics during exposure and after a recovery phase. EGTA was selected for comparison as it has been previously explored as an oral permeation enhancer, and it is an established TJ modulator.
Upon monitoring changes in barrier properties expressed as epithelial integrity decreases after three hours of exposure, four concentrations of penetramax and EGTA that resulted in similar effects were selected for a head-to-head comparison of (i) their time-dependent enhancing properties for permeation of 4 and 10 kDa paracellular markers and (ii) their effect on localization and expression of several TJ proteins as well as their effects on cellular morphology and ultrastructure investigated by immunoblots, confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM), respectively. Lastly, to elucidate the effect of penetramax on paracellular space, STED microscopy was employed to investigate the modulation in bicellular and tricellular junctions.
Despite the similar concentration-dependent effect observed for penetramax and EGTA on modulating the integrity of Caco-2 cell monolayers after three hours of exposure, the kinetics of their effects differed with respect to immediate widening of the paracellular space by penetramax in comparison to a slow and gradual effect of EGTA. Permeation studies revealed that penetramax affects the TJs and transiently widens the paracellular space in a size-selective manner, allowing the permeation of the smaller dextran (4 kDa) to a higher extent than for the 10 kDa dextran. In contrast, EGTA allowed the permeation of 4 and 10 kDa to similar extents, and TEM revealed a severe effect on paracellular space. Immunoblots confirmed that the selectivity of penetramax is related to the alteration of specific TJ protein expression in contrast to the unspecific effects of EGTA. CLSM and TEM confirmed alteration of TJ localization as well as cytoskeletal rearrangement after exposure to penetramax and different from that after exposure to EGTA.
Interestingly, immunoblots upon recovery revealed the alteration of specific TJ protein expression for EGTA, suggesting a de novo protein synthesis of TJs, which resulted in the intracellular localization of TJs, indicating a delayed recovery of the paracellular space in comparison to that after exposure to penetramax. The effect of modulating TJ and cytoskeleton dynamics was also studied upon recovery; therefore, the present thesis addresses the importance of studying the re-epithelization process upon exposure of intestinal epithelium to excipients. Lastly, the potential advantages of employing peptide-based excipients are discussed.
In conclusion, this project showed that functional excipients widen the paracellular space by modulating TJs and the cytoskeleton through different modes of action. This knowledge can be used as a parameter for selecting excipients to improve the therapeutic efficacy of peptide drugs when administered orally.