Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
J Drug Target. 2013 Aug;21(7):662-74. doi: 10.3109/1061186X.2013.796954. Epub 2013 May 22.
Protein delivery vectors can be grouped into two classes, those with specific membrane receptors undergoing conventional endocytosis and cell penetrating peptides (CPP) that have the capacity to cross cell or endosomal membranes. For both forms of vectors, translocation across a membrane is usually an inefficient process. In the current study, a novel vector combining the widely used CPP, Tat and the non-toxic neuronal binding domain of tetanus toxin (fragment C or TTC) was assessed for its capacity to deliver GFP as a test cargo protein to human neural progenitor cells (NPCs). These two functional membrane interacting domains dramatically enhanced internalization of the conjugated cargo protein. Tat-TTC-GFP was found to be bound or internalized at least 83-fold more than Tat-GFP and 33-fold more than TTC-GFP in NPCs by direct fluorimetry, and showed enhanced internalization by quantitative microscopy of 18 - and 14-fold, respectively. This preferential internalization was observed to be specific to neuronal cell types. Photochemical internalization (PCI) was utilized to facilitate escape of the endosome-sequestered proteins. The combined use of the Tat-TTC delivery vector with PCI led to both enhancement of neural cell type specific delivery to an endosomal target, followed by the option of efficient release to the cytosol.
蛋白质递送载体可分为两类，一类是具有特定膜受体的载体，通过常规内吞作用进入细胞；另一类是细胞穿透肽（CPP），能够穿透细胞膜或内体膜。对于这两种载体形式，跨膜转位通常是一个低效的过程。在本研究中，评估了一种新型载体，该载体结合了广泛使用的 CPP Tat 和破伤风毒素的无毒神经元结合结构域（片段 C 或 TTC），以评估其将 GFP 作为测试货物蛋白递送至人神经祖细胞（NPC）的能力。这两个功能膜相互作用结构域显著增强了共轭货物蛋白的内化。通过直接荧光法发现，Tat-TTC-GFP 在 NPC 中的结合或内化至少比 Tat-GFP 高 83 倍，比 TTC-GFP 高 33 倍，通过定量显微镜观察，内化分别增强了 18 倍和 14 倍。这种优先内化被观察到是神经元细胞类型特有的。光化学内吞（PCI）被用于促进内体隔离蛋白的逃逸。Tat-TTC 递药载体与 PCI 的联合使用不仅增强了内体靶标向神经细胞类型的特异性递药，还提供了向细胞质高效释放的选择。
