A Method to Prepare a Bioprobe for Regulatory Science of the Drug Delivery System to the Brain: An Angulin Binder to Modulate Tricellular Tight Junction-Seal.

Epithelium acts as a barrier separating the interior and exterior of the body, and the epithelial and endothelial cells form tight junctions (TJs) by sealing the paracellular space. The blood-brain barrier (BBB) endothelial cells have well-developed TJs and express specific polarized transport systems to tightly control paracellular movements of solutes, ions, and water. Thus, more than 98% of small-molecular-weight drugs cannot pass the BBB. The tricellular TJ (tTJ) is a structure at contacts of three cells. Angulin-1, also known as lipolysis-stimulated lipoprotein receptor (LSR), is one of angulin family and is abundantly expressed in brain endothelial cells, which plays an important role in barrier function of the BBB. The C-terminal domain of a receptor-binding component of Clostridium perfringens iota-toxin (Ib421-664), also named as angubindin-1, binds to its receptors angulin-1 and angulin-3. This angubindin-1 modulates the tTJ barrier and is able to deliver a 16-mer gapmer antisense oligonucleotide (5.3 kDa) without adverse effects. Thus, angulin binders, such as angubindin-1, are useful tools for studying the safety assessment of tTJ-targeted drug delivery and BBB permeability modulation. Here, we provide a protocol for the expression and purification of recombinant angubindin-1 protein as angulin binders, an analysis method for angubindin-1 binding affinity, and a procedure for assessing the effect of modulating tight junction integrity.