Highly cationic cell-penetrating peptides affect the barrier integrity and facilitates mannitol permeation in a human stem cell-based blood-brain barrier model

The Cell-Penetrating Peptide Tat Facilitates Effective Internalization of PSD-95 Inhibitors Into Blood–Brain Barrier Endothelial Cells but less Efficient Permeation Across the Blood–Brain Barrier In Vitro and In Vivo

“…Incubation with T-NR2B9c, T-Tat, T-Tat-N-dimer, and T-Tat-NR2B9c resulted in a similar initial drop in TEER (~50% of the initial value), which leveled out for monolayers incubated with T-NR2B9c and T-Tat-N-dimer, but continued to decrease upon incubation with T-Tat and T-Tat-NR2B9c (Figure 4A). The T-Tat and T-Tat-NR2B9c-mediated drop in TEER did, however, not facilitate the permeation of C 14mannitol, in contrast to another study demonstrating T-Tatmediated C 14 -mannitol permeation across BIONi010-C cell monolayers (Frøslev et al, 2022). The contradictory results may be explained by the fact that the present study was performed in cell culture media supplemented with serum, whereas the earlier study was conducted in a simple HBSS buffer.…”

“…Recent studies have demonstrated that some cationic CPPs modulate the integrity of endothelial (Kristensen et al, 2020b;Frøslev et al, 2022) and epithelial (Lindgren et al, 2004;Diedrichsen et al, 2021) barriers. This suggests potential involvement of the paracellular route in addition to the more generally accepted transcytosis in CPP-mediated drug delivery across the BBB (Hervé et al, 2008;Lim et al, 2015).…”

“…A plausible explanation for these contradictory results is that T-Tat and T-Tat-NR2B9c tend to self-associate and/or adsorb to plasma protein to a greater extent in the cell media than in the simpler HBSS buffer (Figure 1B). This will, in cell culture media, hinder the Tatmoiety from interacting with the plasma membrane of the endothelial cells and eventually result in less dramatic effects on the barrier resistance when compared to studies performed in a simple buffer (Frøslev et al, 2022).…”

“…Thereafter, the cell monolayers were washed twice with 4 °C hHBSS before the plates were placed on ice. The peptide fraction adhering to the plasma membrane, the peptide fraction embedded in the membrane, and the peptide fraction taken up by the cells were separated as earlier described by Frøslev et al (Frøslev et al, 2022) using a modified protocol from Trier et al (Trier et al, 2014). The cell surface-adhered peptide fraction was isolated by the addition of 300 µl of ice-cold 100 µM acetic acid supplemented with 15 μM sodium acetate trihydrate, 10 μM glucose, 1 μM ethylenediaminetetraacetic acid (EGTA), 1.3 μM MgSO 4 heptahydrate, 5 μM KCl, 100 μM NaCl, and 1% BSA (adjusted to pH 3) for 5 min.…”

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

“…Paracellular spaces are aqueous filled channels through which these drugs prefer to diffuse ( Salamat-Miller and Johnston, 2005 ; Patel and Misra, 2011 ). Several studies have reported that some CPPs can also promote permeation of various cellular barriers through paracellular route both in vitro and in vivo ( Ohtake et al, 2002 ; Bai et al, 2008 ; Kristensen and Nielsen, 2016a ; Kristensen and Nielsen, 2016b ; Kristensen et al, 2020 ; Diedrichsen et al, 2021 ; Ragupathy et al, 2021 ; Frøslev et al, 2022 ). This could be either due to the high local concentration of the CPPs, influencing the dynamics of the tight junction proteins or due to cell-penetrating tendency of CPPs, targeting intracellular proteins which are involved in regulation of opening and closing of tight junctions ( Taverner et al, 2015 ; Kristensen and Nielsen, 2016a ).…”