Investigating the Mechanism of Drug Carrier Transport through Endothelial Cell Barriers

 性状（由申请方提供）：内衬于血管内部的内皮细胞阻碍药物载体从血流转运至患病组织。为了克服这一障碍，药物载体可以靶向介导穿过这一屏障的转胞吞作用的细胞表面受体。然而，药物载体在运输后从屏障分离并通过下层细胞外基质（ECM）迁移的机制仍然未知。因此，这项工作的总体目标是检查这些方面，以更好地了解参与调节和调节内皮屏障功能的机制。在这样做的过程中，这项工作将指导翻译药物输送治疗的疾病，需要跨内皮运输。作为临床相关的应用实例，该提议将在靶向细胞间粘附分子-1（ICAM-1）并携带酶货物（酸性鞘磷脂酶，ASM）的聚合物纳米载体用于治疗A-B型尼曼-皮克病（一种由溶酶体ASM缺乏引起的溶酶体贮积症，其导致严重的肺和脑功能障碍）的情况下探索这些问题。具体而言，ICAM-1是研究药物载体转运机制的有趣且相关的靶标，因为其在病理条件下在内皮上高度表达，并且促进天然（白细胞）和人工（ICAM-1靶向药物载体）配体穿过内皮屏障的转运。与显示ICAM-1和基质金属蛋白酶（MMP），特别是MMP-9和MMP-2之间相互作用的文献一致，初步结果表明，载体与ICAM-1结合导致MMP-9和MMP-2从内皮细胞分泌，并且MMP-9有助于ICAM-1裂解。因此，假设MMP在载体与ICAM-1结合时释放导致（a）ICAM-1裂解，允许载体在胞吞转运后从屏障脱离，和（B）ECM降解，允许载体渗透到组织中。为了研究这些假设，本提案在细胞培养和动物模型中检查MMP-9和MMP-2在（1）ICAM-1切割、（2）载体跨内皮屏障转运和（3）ECM降解中对载体渗透的贡献。首先，ELISA和Western印迹将用于评估MMP分泌和ICAM-1切割，以及检查MMP介导的ICAM-1释放和载体生物分布之间的相关性。第二，放射性同位素示踪将用于检查通过ICAM-1裂解的载体跨内皮屏障的转运。第三，将使用明胶酶谱法、荧光显微镜、免疫组织化学和TEM成像评估ECM降解和载体渗透。在每种情况下，将使用MMP-9和/或MMP-2缺陷的细胞培养物和动物模型来鉴定它们在这些过程中的作用。通过完成这些目标，这项工作将改善许多需要通过不可渗透的内皮屏障转运的药物治疗。这项研究也将大大有助于 将PI培训为独立研究者，从而使未来的研究能够促进生物医学技术的发展。

 DESCRIPTION (provided by applicant): Endothelial cells lining the interior of blood vessels hinder transport of drug carriers from the bloodstream into diseased tissues. In order to overcome this obstacle, drug carriers can be targeted to cell-surface receptors that mediate transcytosis across this barrier. However, the mechanism by which drug carriers detach from the barrier after transport and migrate through the underlying extracellular matrix (ECM) is still unknown. Therefore, the overall goal of this work is to examine these aspects for better understanding of the mechanisms involved in regulating and modulating endothelial barrier function. In doing so, this work will guide translational drug delivery for treatment of diseases where transendothelial transport is required. As a clinically-relevant example of an application, this proposal will explore these questions in the case of polymer nanocarriers targeted to intercellular adhesion molecule-1 (ICAM-1) and carrying an enzyme cargo (acid sphingomyelinase, ASM) for treatment of type A-B Niemann-Pick Disease, a lysosomal storage disorder caused by deficiency of lysosomal ASM, which results in severe pulmonary and brain dysfunction. Specifically, ICAM-1 is an interesting and relevant target to study the mechanism of drug carrier transport because it is highly expressed on the endothelium during pathological conditions, and facilitates transport of natural (leukocytes) and artificial (ICAM-1-targeted drug carriers) ligands across endothelial barriers. In agreement with literature showing the interplay between ICAM-1 and matrix metalloproteinases (MMPs), particularly MMP-9 and MMP-2, preliminary results indicate that carrier binding to ICAM-1 causes MMP-9 and MMP-2 secretion from endothelial cells, and MMP-9 contributes to ICAM-1 cleavage. Therefore, it is hypothesized that MMP release upon carrier binding to ICAM-1 results in (a) ICAM-1 cleavage, allowing carrier detachment from the barrier after transcytosis, and (b) ECM degradation, allowing carrier penetration into tissues. To investigate these hypotheses, this proposal examines the contribution of MMP-9 and MMP-2 in (1) ICAM-1 cleavage, (2) carrier transport across endothelial barriers, and (3) ECM degradation for carrier penetration in both cell culture and animal models. First, ELISA and Western blotting will be used to assess MMP secretion and ICAM-1 cleavage, as well as examine the correlation between MMP-mediated release of ICAM-1 and carrier biodistribution. Second, radioisotope tracing will be used to examine carrier transport across endothelial barriers via ICAM-1 cleavage. Third, ECM degradation and carrier penetration will be assessed using gelatin zymography, fluorescence microscopy, immunohistochemistry, and TEM imaging. In each case, cell culture and animal models deficient in MMP-9 and/or MMP-2 will be used to identify their role in these processes. Through completion of these aims, this work stands to improve a number of drug therapies requiring transport across impermeable endothelial barriers. This research will also significantly contribute to training the PI as an independent investigator, thereby enabling future research for the advancement of biomedical technologies.