Screening for effective cell-penetrating peptides with minimal impact on epithelial cells and gut commensals in vitro

Abstract

One of the biggest challenges for oral drug absorption is the epithelial barrier of the gastrointestinal tract. The use of cell-penetrating peptides (CPPs) to modulate the epithelial barrier function is known to be an effective strategy to improve drug absorption and bioavailability. In this study we compare side-by-side, 9 most promising CPPs to study their cytotoxicity (Cytotox Red dye staining) and cell viability (AlamarBlue staining) on epithelial cells and their effects on paracellular permeability of the intestinal barrier in vitro in a differentiated Caco-2 epithelial monolayer model. The data revealed that 4 out of 9 well-studied CPPs significantly improved Caco-2 paracellular permeability without compromising on cellular health. To assess the impact of CPPs on the human microbiota we studied the antimicrobial effects of the 4 effective CPPs from our permeation studies against 10 representative strains of the gut microbiota in vitro using microbroth dilution. Our data revealed that these 4 CPPs affected the growth of almost all tested commensal strains. Interestingly, we found that two synthetic CPPs (Shuffle and Penetramax) outperformed all the other CPPs in their ability to increase intestinal paracellular permeability at 50 µM and had only a small to moderate effect on the tested gut commensal strains. Based on these data Shuffle and Penetramax represent relevant CPPs to be further characterized in vivo for safe delivery of poorly absorbed therapeutics while minimizing negative impacts on the gut microbiota.

Track‐Etched Membranes for Drug Pharmaceutical Research

“…Unlike membranes with a sponge-like structure, these membranes do not let cells sink into the material or adhere to the pores but grow where they are supposed to: on the extremely smooth surface of the microporous membrane. Cells grown on these membranes have a basal and apical side, mimicking in vivo conditions, enabling cell polarization and promoting adequate cell differentiation [13,23]. Track-etched membranes are widely used to study drug permeability and transport [13].…”

“…Cells grown on these membranes have a basal and apical side, mimicking in vivo conditions, enabling cell polarization and promoting adequate cell differentiation [13,23]. Track-etched membranes are widely used to study drug permeability and transport [13]. Moreover, they are suitable to perform migration and invasion studies in the context of cancer and immunotherapy drug research [24].…”

“…The use of cell culture inserts offers the possibility of adaptation to dynamic cell culture conditions [44,45] and co-cultivation of cells on both sides of the membrane [46]. In addition, drug permeability tests can be performed as solutions can be collected from the apical and basolateral compartments [13]. The ability to remove medium from the apical compartment provides an excellent option for culturing cells at the air-liquid interface, a requirement for maintaining important phenotypes in many cells.…”

“…The intestinal epithelial lining is important for the oral delivery of drugs. In a recent study, Caco-2 cells grown on semi-permeable cell culture inserts were used to model the intestinal epithelial monolayer to assess the effect of several cell-penetrating peptides in barrier integrity [13]. The use of cell-penetrating peptides is a strategy to improve drug uptake by promoting permeation in the context of drug delivery.…”

“…Numerous state-of-the-art technologies in cell culture contain tracketched membranes as integral and critical components of the system. Normally, the membrane is the support on which the cells adhere and grow [12][13][14][15]. In this review, we provide examples of relevant in vitro models where tracketched membranes are integrated.…”