细胞穿透肽(CPPs)Tat和penetratin经常被用作跨血脑屏障(BBB)的药物递送穿梭。CPPs通常用荧光团标记用于分析目的,5(6)-羧基四甲基罗丹明(TAMRA)是一种流行的选择。然而,当与单独的CPP相比时,TAMRA标记影响所得荧光团-CPP构建体的物理化学性质。硒蛋氨酸(MSe)可以作为替代标签引入,which,由于其体积小和氨基酸性质,可能导致肽理化性质的最小改变。我们与这项研究进行了比较,头对头,Tat和penetratin的MSe和TAMRA标记对其理化性质的影响,并研究了其对脑毛细血管内皮细胞(BCEC)模型的影响。当与MSe标记相比时,TAMRA标记积极影响肽粘附到细胞膜以及内化到BCEC中的能力。与TAMRA标记Tat相比,TAMRA标记对BCECs的毒性更高,而MSe标记不影响细胞活力。TAMRA和MSe标记penetratin都降低了BCEC单层的屏障完整性,但不能改善细胞旁标记物14C-甘露醇的转运。总之,Tat和penetratin的MSe标签对这些CPPs的物理化学性质及其对BCECs的影响增加了最小的改变,从而代表了用于肽定量目的的TAMRA的优选替代方案。
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 physicochemical properties of the resulting fluorophore-CPP construct when compared to the CPP alone. Selenomethionine (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.