Transport of cationized anti-tetanus Fab ' 2 fragments across an in vitro blood-brain barrier model: Involvement of the transcytosis pathway

Tetanus neurotoxin reaches the CNS by axonal retrograde transport and thus becomes inaccessible to current treatments, A possible strategy to improve current therapy for tetanus disease would be the vectorization of Fab'2 fra gments, allowing their delivery into the CNS. The purpose of this study was to investigate whether after cationization anti-tetanus Fab'2 fragments ar e able to cross the blood-brain barrier, the first obstacle to CNS delivery . We used primary cocultures of bovine brain capillary endothelial cells an d newborn rat astrocytes as an in vitro model to study the binding and tran sport of cationized Fab'2 (cFab'2) fragments across the brain endothelium, We first show that cationization does not alter Fab'2 affinity for tetanus toxin. Then we demonstrate that after cationization Fab'2! fragments are ab le to bind to the negative charges on the surface of endothelial cells and subsequently to be transported across the endothelial cell monolayer withou t any modification of affinity. Finally, using fluorescence microscopy, we show that cFab'2 fragments are transported through endocytotic vesicles. Th e present study demonstrates that cationization allows Fab'2 directed again st tetanus toxin to be transported through brain endothelium by adsorptive- mediated transcytosis, We suggest that this vectorization way could be a pr omising delivery strategy for carrying anti-tetanic immunoglobulin fragment s across the blood-brain barrier to improve tetanus treatment.