Development of a Neuroprotective Erythropoietin Modified with a Novel Carrier for the Blood–Brain Barrier

Extremely high doses of erythropoietin (EPO) has been used for neuroprotection in ischemia–reperfusion brain injury to deliver sufficient amounts of EPO across the blood–brain barrier (BBB); however, harmful outcomes were observed afterward. We aimed to test the ability of HBHAc (heparin-binding haemagglutinin adhesion c), an intracellular delivery peptide for macromolecules, as an EPO carrier across the BBB. The cellular internalization and transcytosis ability of HBHAc-modified EPO (EPO-HBHAc) were evaluated in bEnd.3 cells and in the bEnd.3/CTX TNA2 co-culture BBB model, respectively. Subsequently, the NMDA-induced-toxicity model and ischemia–reperfusion rat model were used to understand the neuronal protective activity of EPO-HBHAc. The biodistribution of EPO-HBHAc was demonstrated in rats by the quantification of EPO-HBHAc in the brain, plasma, and organs by ELISA. Our results demonstrate that EPO-HBHAc exhibited significantly higher cellular internalization in dose- and time-dependent manners and better transcytosis ability than EPO. In addition, the transported EPO-HBHAc in the co-culture transwell system maintained the neuronal protective activity when primary rat cortical neurons underwent NMDA-induced toxicity. The calculated cerebral infarction area of rats treated with EPO-HBHAc was significantly reduced compared to that of rats treated with EPO (29.9 ± 7.0% vs 48.9 ± 7.9%) 24 h after occlusion in 3VO rat experiments. Moreover, the EPO amount in both CSF and damaged cortex from the EPO-HBHAc group was 4.0-fold and 3.0-fold higher than the EPO group, respectively. These results suggest that HBHAc would be a favorable tool for EPO brain delivery and would further extend the clinical applications of EPO in neuroprotection.