Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, MD 20742, USA.
J Control Release. 2012 Feb 10;157(3):485-92. doi: 10.1016/j.jconrel.2011.09.067. Epub 2011 Sep 16.
Delivery of drugs into the endothelium by nanocarriers targeted to endothelial determinants may improve treatment of vascular maladies. This is the case for intercellular adhesion molecule 1 (ICAM-1), a glycoprotein overexpressed on endothelial cells (ECs) in many pathologies. ICAM-1-targeted nanocarriers bind to and are internalized by ECs via a non-classical pathway, CAM-mediated endocytosis. In this work we studied the effects of endothelial adaptation to physiological flow on the endocytosis of model polymer nanocarriers targeted to ICAM-1 (anti-ICAM/NCs, 180 nm diameter). Culturing established endothelial-like cells (EAhy926 cells) and primary human umbilical vein ECs (HUVECs) under 4 dyn/cm(2) laminar shear stress for 24 h resulted in flow adaptation: cell elongation and formation of actin stress fibers aligned to the flow direction. Fluorescence microscopy showed that flow-adapted cells internalized anti-ICAM/NCs under flow, although at slower rate versus non flow-adapted cells under static incubation (35% reduction). Uptake was inhibited by amiloride, whereas marginally affected by filipin and cadaverine, implicating that CAM-endocytosis accounts for anti-ICAM/NC uptake under flow. Internalization under flow was more modestly affected by inhibiting protein kinase C, which regulates actin remodeling during CAM-endocytosis. Actin recruitment to stress fibers that maintain the cell shape under flow may delay uptake of anti-ICAM/NCs under this condition by interfering with actin reorganization needed for CAM-endocytosis. Electron microscopy revealed somewhat slow, yet effective endocytosis of anti-ICAM/NCs by pulmonary endothelium after i.v. injection in mice, similar to that of flow-adapted cell cultures: ~40% (30 min) and 80% (3 h) internalization. Similar to cell culture data, uptake was slightly faster in capillaries with lower shear stress. Further, LPS treatment accelerated internalization of anti-ICAM/NCs in mice. Therefore, regulation of endocytosis of ICAM-1-targeted nanocarriers by flow and endothelial status may modulate drug delivery into ECs exposed to different physiological (capillaries vs. arterioles/venules) or pathological (ischemia, inflammation) levels and patterns of blood flow.
通过靶向内皮细胞决定因素的纳米载体将药物递送至内皮细胞，可能有助于改善血管疾病的治疗效果。细胞间黏附分子 1（ICAM-1）就是这种情况，它是许多病理情况下内皮细胞（EC）过度表达的糖蛋白。靶向 ICAM-1 的纳米载体通过非经典途径，即细胞黏附分子（CAM）介导的内吞作用与 EC 结合并被其内化。在这项工作中，我们研究了内皮细胞对生理流动的适应对内吞靶向 ICAM-1 的模型聚合物纳米载体（抗 ICAM/NC，180nm 直径）的影响。将已建立的内皮样细胞（EAhy926 细胞）和原代人脐静脉内皮细胞（HUVEC）在 4 dyn/cm2层流剪切力下培养 24 小时可导致细胞适应流动：细胞伸长并形成沿流动方向排列的肌动蛋白应力纤维。荧光显微镜显示，在流动条件下，经流动适应的细胞会内化抗 ICAM/NC，尽管与静态孵育下未经流动适应的细胞相比，内化速度较慢（35%的降低）。阿米洛利抑制了摄取，而 Filipin 和 Cadaverine 则轻微影响摄取，提示 CAM 内吞作用解释了流动条件下抗 ICAM/NC 的摄取。蛋白激酶 C 的抑制对内吞作用的影响较小，蛋白激酶 C 调节 CAM 内吞作用过程中的肌动蛋白重塑。在流动条件下，肌动蛋白募集到维持细胞形状的应力纤维可能会通过干扰 CAM 内吞作用所需的肌动蛋白重排来延迟抗 ICAM/NC 的摄取。电子显微镜显示，在静脉注射后，小鼠肺内皮细胞对静脉内注射的抗 ICAM/NC 的内吞作用较慢，但仍有效，类似于经流动适应的细胞培养物：~40%（30 分钟）和 80%（3 小时）的内化。与细胞培养数据类似，剪切应力较低的毛细血管内的摄取速度稍快。此外，LPS 处理可加速小鼠抗 ICAM/NC 的内化。因此，流动和内皮状态对内靶向纳米载体摄取的调节可能会调节暴露于不同生理（毛细血管与小动脉/小静脉）或病理（缺血、炎症）血流水平和模式的内皮细胞中的药物输送。
