Rosenberg Michal, Zilony Neta, Shefi Orit, Segal Ester
Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology.
Faculty of Engineering, Bar-Ilan University; Bar-Ilan Institute of Nanotechnologies and Advanced Materials.
J Vis Exp. 2019 Jan 25(143). doi: 10.3791/58982.
Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.
尽管神经生长因子（NGF）在治疗神经退行性疾病方面具有巨大潜力，但其治疗给药却面临重大挑战，因为该蛋白质因其化学性质无法穿过血脑屏障，因此需要长期向脑部给药才能产生生物学效应。这项工作描述了纳米结构的多孔硅（PSi）薄膜的制备，作为NGF的可降解载体用于持续递送这种敏感蛋白质。PSi载体经过特殊设计，以获得高负载效率并在四周内持续释放NGF，同时保持其生物活性。通过检查其诱导PC12细胞和离体背根神经节（DRG）神经元分化和生长的能力，在体外研究了NGF-PSi载体作为NGF递送系统的行为。评估了在纯PSi载体和负载NGF的PSi载体存在下的细胞活力。将从PSi载体释放的NGF的生物活性与重复游离NGF给药的传统治疗方法进行比较。通过测量分化细胞的三个不同形态学参数来分析和表征PC12细胞分化；（i）从细胞体伸出的神经突数量（ii）神经突总长度和（iii）分支点数量。用NGF-PSi载体处理的PC12细胞在整个释放期都表现出深刻的分化。此外，用NGF-PSi载体培养的DRG神经元细胞显示出广泛的神经突起始，类似于用重复游离NGF给药处理的神经元。所研究的可调谐载体证明了用于NGF释放的长期植入物对神经退行性疾病具有治疗潜力。
