Selenomethionine as alternative label to the fluorophore TAMRA when exploiting cell-penetrating peptides as blood-brain barrier shuttles to better mimic the physicochemical properties of the non-labelled peptides

The cell-penetrating peptides (CPPs) Tat and penetratin are frequently explored as shuttles for drug delivery across the blood-brain barrier (BBB). CPPs are often labelled with fluorophores for analytical purposes, with 5(6)-carboxytetramethylrhodamine (TAMRA) being a popular choice. However, TAMRA labelling affects the physi-cochemical properties of the resulting fluorophore-CPP construct when compared to the CPP alone. Selenome-thionine (MSe) may be introduced as alternative label, which, due to its small size and amino acid nature, likely results in minimal alterations of the peptide physicochemical properties. With this study we compared, head-to -head, the effect of MSe and TAMRA labelling of Tat and penetratin with respect to their physicochemical properties, and investigated effects hereof on brain capillary endothelial cell (BCEC) models. TAMRA labelling positively affected the ability of the peptides to adhere to the cell membranes as well being internalized into the BCECs when compared to MSe labelling. TAMRA labelling of penetratin added toxicity to the BCECs to a higher extent than TAMRA labelling of Tat, whereas MSe labelling did not affect the cellular viability. Both TAMRA and MSe labelling of penetratin decreased the barrier integrity of BCEC monolayers, but not to an extent that improved transport of the paracellular marker 14C-mannitol. In conclusion, MSe labelling of Tat and penetratin adds minimal alterations to the physicochemical properties of these CPPs and their resulting effects on BCECs, and thereby represents a preferred alternative to TAMRA for peptide quantification purposes.

Automated summary

The study compared the effect of labelling Tat and penetratin with 5(6)-carboxytetramethylrhodamine (TAMRA) and selenomethionine (MSe) on their physicochemical properties and on brain capillary endothelial cell (BCEC) models. TAMRA labelling increased the adherence and internalization of the peptides in BCECs compared to MSe labelling. However, TAMRA labelling of penetratin had a higher toxicity effect on the BCECs compared to TAMRA labelling of Tat, while MSe labelling did not affect cellular viability. Both TAMRA and MSe labelling decreased the barrier integrity of BCEC monolayers, but did not improve transport of the paracellular marker 14C-mannitol. Therefore, MSe labelling is preferred over TAMRA for quantification of peptides due to minimal alterations in physicochemical properties and effects on BCECs.