PDF(Pubmed)
Abstract：
Current strategies to identify ligands for brain delivery select candidates based on preferential binding to cell-membrane components (CMC) on brain endothelial cells (EC). However, such strategies generate ligands with inherent brain specificity limitations, as the CMC (e.g., the transferrin receptor TfR1) are also significantly expressed on peripheral EC. Therefore, novel strategies are required to identify molecules allowing increased specificity of therapy brain delivery. Here, we demonstrate that, while individual CMC are shared between brain EC and peripheral EC, their endocytic internalization rate is markedly different. Such differential endocytic rate may be harnessed to identify molecular tags for brain targeting based on their selective retention on the surface of brain EC, thereby generating \'artificial\' targets specifically on the brain vasculature. By quantifying the retention of labelled proteins on the cell membrane, we measured the general endocytic rate of primary brain EC to be less than half that of primary peripheral (liver and lung) EC. In addition, through bio-panning of phage-displayed peptide libraries, we unbiasedly probed the endocytic rate of individual CMC of liver, lung and brain endothelial cells. We identified phage-displayed peptides which bind to CMC common to all three endothelia phenotypes, but which are preferentially endocytosed into peripheral EC, resulting in selective retention on the surface of brain EC. Furthermore, we demonstrate that the synthesized free-form peptides are capable of generating artificial cell-surface targets for the intracellular delivery of model proteins into brain EC with increasing specificity over time. The developed identification paradigm, therefore, demonstrates that the lower endocytic rate of individual CMC on brain EC can be harnessed to identify peptides capable of generating \'artificial\' targets for the selective delivery of proteins into the brain vasculature. In addition, our approach identifies brain-targeting peptides which would have been overlooked by conventional identification strategies, thereby increasing the repertoire of candidates to achieve specific therapy brain delivery.
摘要：
识别用于脑递送的配体的当前策略基于与脑内皮细胞(EC)上的细胞膜组分(CMC)的优先结合来选择候选物。然而,这样的策略产生具有固有脑特异性限制的配体，作为CMC(例如，转铁蛋白受体TfR1）在外周EC上也显着表达。因此,需要新的策略来鉴定允许增加治疗脑递送特异性的分子.这里,我们证明，虽然个体CMC在脑EC和外周EC之间共享，它们的内吞内化速率明显不同。可以利用这种差异内吞速率来基于其在脑EC表面的选择性保留来识别用于脑靶向的分子标签。从而在脑血管系统上产生“人造”目标。通过定量标记蛋白质在细胞膜上的保留，我们测量的原发性脑EC的一般内吞率小于原发性外周（肝和肺）EC的一半。此外,通过噬菌体展示肽文库的生物淘选，我们不偏不倚地探测了肝脏单个CMC的内吞率，肺和脑内皮细胞。我们鉴定了与所有三种内皮表型共同的CMC结合的噬菌体展示肽，但它们优先内吞进入外周EC，导致选择性保留在脑EC表面。此外，我们证明,合成的游离形式肽能够产生人工细胞表面靶标,用于将模型蛋白细胞内递送到脑EC中,随着时间的推移特异性增加.发达的识别范式，因此,表明，可以利用单个CMC对脑EC的较低内吞速率来鉴定能够产生“人工”靶标的肽，以将蛋白质选择性递送到脑血管系统中。此外,我们的方法识别了常规识别策略会忽略的脑靶向肽，从而增加候选人的库，以实现特定的治疗脑输送。
