The improved blood-brain barrier permeability of endomorphin-1 using the cell-penetrating peptide synB3 with three different linkages.

Endomorphins, although they have high analgesic activity and few undesirable side effects, are not in clinical use because of the blood-brain barrier (BBB). One promising solution is to use cell-penetrating peptides (CPPs). CPPs have the ability to translocate cell membranes and have been successfully applied for delivery of therapeutic molecules across the BBB. However, little is known about the transport efficiency of different conjugation strategies between cargo and CPPs. In this study, endomorphin-1 (EM-1) was conjugated with SynB3, an efficient CPP-carrier, via amide, maleimide and disulfide linkages. The delivery efficiency of three linkers was compared in terms of pharmacodynamics and in vitro metabolic stability. Near-infrared fluorescent and fluorescent microscopy experiments were applied to detect the brain uptake and distribution of CPP delivery qualitatively and quantitatively. After the most successful linkage was screened out, the further mechanisms were discussed. We concluded that compared with the other two linkages, the disulfide bond was the most efficient linkage to deliver EM-1 across the BBB and confirmed that it could be reduced at physiological conditions in the brain and release its active form. These findings indicate that for those who need to release a free drug in the brain and maintain activity, a disulfide bond might be the most efficient linkage across the BBB.