Department of Biomedicine, Faculty of Health, Aarhus University, Aarhus, Denmark.
Department of Health Technology, Technical University of Denmark, Lyngby, Denmark.
PLoS One. 2021 Apr 2;16(4):e0249686. doi: 10.1371/journal.pone.0249686. eCollection 2021.
The blood-brain barrier (BBB) is one of the main obstacles for therapies targeting brain diseases. Most macromolecules fail to pass the tight BBB, formed by brain endothelial cells interlinked by tight junctions. A wide range of small, lipid-soluble molecules can enter the brain parenchyma via diffusion, whereas macromolecules have to transcytose via vesicular transport. Vesicular transport can thus be utilized as a strategy to deliver brain therapies. By conjugating BBB targeting antibodies and peptides to therapeutic molecules or nanoparticles, it is possible to increase uptake into the brain. Previously, the synthetic peptide GYR and a peptide derived from melanotransferrin (MTfp) have been suggested as candidates for mediating transcytosis in brain endothelial cells (BECs). Here we study uptake, intracellular trafficking, and translocation of these two peptides in BECs. The peptides were synthesized, and binding studies to purified endocytic receptors were performed using surface plasmon resonance. Furthermore, the peptides were conjugated to a fluorophore allowing for live-cell imaging studies of their uptake into murine brain endothelial cells. Both peptides bound to low-density lipoprotein receptor-related protein 1 (LRP-1) and the human transferrin receptor, while lower affinity was observed against the murine transferrin receptor. The MTfp showed a higher binding affinity to all receptors when compared to the GYR peptide. The peptides were internalized by the bEnd.3 mouse endothelial cells within 30 min of incubation and frequently co-localized with endo-lysosomal vesicles. Moreover, our in vitro Transwell translocation experiments confirmed that GYR was able to cross the murine barrier and indicated the successful translocation of MTfp. Thus, despite binding to endocytic receptors with different affinities, both peptides are able to transcytose across the murine BECs.
血脑屏障 (BBB) 是针对脑部疾病的治疗方法的主要障碍之一。大多数大分子无法通过由紧密连接相连的脑内皮细胞形成的紧密 BBB。大量的小、脂溶性分子可以通过扩散进入脑实质，而大分子则必须通过胞吞作用进行转运。因此，胞吞作用可以作为一种向大脑输送治疗方法的策略。通过将针对 BBB 的抗体和肽与治疗分子或纳米颗粒缀合，可以增加进入大脑的摄取量。以前，合成肽 GYR 和来源于转铁蛋白 (MTfp) 的肽被认为是介导脑内皮细胞 (BEC) 胞吞作用的候选物。在这里，我们研究了这两种肽在 BEC 中的摄取、细胞内运输和转位。合成了这些肽，并使用表面等离子体共振进行了与纯化的内吞受体的结合研究。此外，将这些肽与荧光团缀合，以允许对其进入小鼠脑内皮细胞的摄取进行活细胞成像研究。两种肽都与低密度脂蛋白受体相关蛋白 1 (LRP-1) 和人转铁蛋白受体结合，而对鼠转铁蛋白受体的亲和力较低。与 GYR 肽相比，MTfp 对所有受体的结合亲和力更高。肽在孵育 30 分钟内被 bEnd.3 小鼠内皮细胞内化，并且经常与内溶酶体囊泡共定位。此外，我们的体外 Transwell 转位实验证实 GYR 能够穿过小鼠屏障，并表明 MTfp 成功转位。因此，尽管与内吞受体的亲和力不同，但这两种肽都能够穿过小鼠 BEC 进行胞吞。
