Wang Ting-Yi, Bruggeman Kiara A F, Sheean Rebecca K, Turner Bradley J, Nisbet David R, Parish Clare L
From the Florey Institute of Neuroscience & Mental Health, The University of Melbourne, Parkville, Australia, 3010 and.
the Research School of Engineering, The Australian National University, Canberra, Australia, 0200.
J Biol Chem. 2014 May 23;289(21):15044-51. doi: 10.1074/jbc.M113.537381. Epub 2014 Apr 3.
Various engineering applications have been utilized to deliver molecules and compounds in both innate and biological settings. In the context of biological applications, the timely delivery of molecules can be critical for cellular and organ function. As such, previous studies have demonstrated the superiority of long-term protein delivery, by way of protein tethering onto bioengineered scaffolds, compared with conventional delivery of soluble protein in vitro and in vivo. Despite such benefits little knowledge exists regarding the stability, release kinetics, longevity, activation of intracellular pathway, and functionality of these proteins over time. By way of example, here we examined the stability, degradation and functionality of a protein, glial-derived neurotrophic factor (GDNF), which is known to influence neuronal survival, differentiation, and neurite morphogenesis. Enzyme-linked immunosorbent assays (ELISA) revealed that GDNF, covalently tethered onto polycaprolactone (PCL) electrospun nanofibrous scaffolds, remained present on the scaffold surface for 120 days, with no evidence of protein leaching or degradation. The tethered GDNF protein remained functional and capable of activating downstream signaling cascades, as revealed by its capacity to phosphorylate intracellular Erk in a neural cell line. Furthermore, immobilization of GDNF protein promoted cell survival and differentiation in culture at both 3 and 7 days, further validating prolonged functionality of the protein, well beyond the minutes to hours timeframe observed for soluble proteins under the same culture conditions. This study provides important evidence of the stability and functionality kinetics of tethered molecules.
在天然和生物环境中，各种工程应用已被用于递送分子和化合物。在生物应用的背景下，分子的及时递送对于细胞和器官功能可能至关重要。因此，先前的研究表明，与体外和体内可溶性蛋白质的传统递送相比，通过将蛋白质拴系到生物工程支架上进行长期蛋白质递送具有优越性。尽管有这些好处，但对于这些蛋白质的稳定性、释放动力学、寿命、细胞内途径的激活以及随时间的功能，人们知之甚少。例如，在这里我们研究了一种蛋白质——胶质细胞源性神经营养因子（GDNF）的稳定性、降解和功能，已知该因子会影响神经元的存活、分化和神经突形态发生。酶联免疫吸附测定（ELISA）显示，共价拴系在聚己内酯（PCL）电纺纳米纤维支架上的GDNF在支架表面保留了120天，没有蛋白质浸出或降解的迹象。拴系的GDNF蛋白仍然具有功能，并且能够激活下游信号级联反应，这一点通过其在神经细胞系中磷酸化细胞内Erk的能力得以揭示。此外，GDNF蛋白的固定在3天和7天时均促进了培养物中的细胞存活和分化，进一步验证了该蛋白的延长功能，远远超过在相同培养条件下可溶性蛋白质所观察到的几分钟到几小时的时间范围。这项研究为拴系分子的稳定性和功能动力学提供了重要证据。
