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.

中文翻译：

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开发一种用新型血脑屏障载体修饰的神经保护性促红细胞生成素。


极高剂量的促红细胞生成素 (EPO) 已用于缺血再灌注脑损伤的神经保护，以输送足够量的 EPO 穿过血脑屏障 (BBB)；然而，随后观察到了有害的结果。我们的目的是测试 HBHAc（肝素结合血凝素粘附 c）（一种大分子细胞内递送肽）作为跨 BBB 的 EPO 载体的能力。分别在 bEnd.3 细胞和 bEnd.3/CTX TNA2 共培养 BBB 模型中评估 HBHAc 修饰的 EPO (EPO-HBHAc) 的细胞内化和转胞吞能力。随后，利用NMDA诱导的毒性模型和缺血再灌注大鼠模型来了解EPO-HBHAc的神经元保护活性。通过 ELISA 对大脑、血浆和器官中的 EPO-HBHAc 进行定量，证明了 EPO-HBHAc 在大鼠体内的生物分布。我们的结果表明，与 EPO 相比，EPO-HBHAc 以剂量和时间依赖性方式表现出显着更高的细胞内化以及更好的转胞吞能力。此外，当原代大鼠皮质神经元遭受 NMDA 诱导的毒性时，共培养 Transwell 系统中转运的 EPO-HBHAc 仍保持神经元保护活性。在 3VO 大鼠实验中，闭塞后 24 小时，用 EPO-HBHAc 治疗的大鼠的计算脑梗塞面积与用 EPO 治疗的大鼠相比显着减少（29.9 ± 7.0% vs 48.9 ± 7.9%）。此外，EPO-HBHAc组的脑脊液和受损皮质中的EPO含量分别是EPO组的4.0倍和3.0倍。这些结果表明，HBHAc 将成为 EPO 大脑递送的有利工具，并将进一步扩展 EPO 在神经保护方面的临床应用。