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.

Study of the intracellular delivery mechanism of a pH-sensitive peptide modified with enhanced green fluorescent protein.

The targeted delivery of therapeutic agents is a promising approach to enhance the efficacy and reduce the toxicity of cancer treatments. Understanding the intracellular endocytic mechanisms of a cell penetrating peptide (CPP) in an acidic environment is important for targeted delivery of macromolecules to tumours. In this study, we constructed a pH-sensitive CPP-based delivery system for the intracellular delivery of macromolecules. A pH-sensitive CPP, HBHAc, was fused with a model protein, enhanced green fluorescent protein (EGFP), through recombinant DNA technology. We found that is essential that negatively charged proteoglycans on the cell surface interact with HBHAc-EGFP prior to the cellular uptake of HBHAc-EGFP. The uptake was significantly restricted at 4 °C under pH conditions of both 6.5 and 7.5. The increased positive charge of HBHAc-EGFP under the acidic condition leads to a pH-dependent cellular uptake, and we observed that the internalisation of HBHAc-EGFP was significantly higher at pH 6.5 than at pH 7.5 (p < .05). Thus, with pH-sensitive activity, HBHAc is expected to improve tumour-targeted intracellular protein delivery. Moreover, our findings provide a new insight that the endocytic pathway may change under different pH conditions and suggest that this unique phenomenon benefits pH-sensitive drug delivery for tumour therapy.