Investigating the Mechanism of Drug Carrier Transport through Endothelial Cell Barriers

研究药物载体通过内皮细胞屏障运输的机制

• 批准号: 8909416
• 负责人: Rachel Lee Manthe
• 金额: $ 2.95万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8909416
• 关键词: Adhesions; Agreement; Animal Model; Binding; Biodistribution; Biology; Biomedical Technology; Blood; Blood Circulation; Blood Vessels; Brain; Caveolae; Cell Culture Techniques; Cell Line; Cell Surface Receptors; Cell physiology; Cell surface; Cells; Clathrin; Cleaved cell; Coupled; Development; Disease; Drug Carriers; Drug Delivery Systems; Drug Transport; Endopeptidases; Endothelial Cells; Endothelium; Enzyme-Linked Immunosorbent Assay; Enzymes; Extracellular Domain; Extracellular Matrix; Extracellular Matrix Degradation; Family; Fluorescence Microscopy; Functional disorder; Gelatin Zymography; Gelatinase A; Gelatinase B; Gelatinases; Goals; Healthcare; Human; Image; Immunohistochemistry; In Vitro; Integral Membrane Protein; Intercellular Junctions; Intercellular adhesion molecule 1; Kinetics; Knock-out; Knowledge; Lead; Leukocytes; Ligands; Literature; Lung; Matrix Metalloproteinases; Mediating; Mission; Modeling; Mus; National Heart, Lung, and Blood Institute; Niemann-Pick Diseases; Penetration; Pharmacotherapy; Polymers; Process; Radioisotopes; Recombinants; Regulation; Research; Research Personnel; Role; Side; Signal Transduction; Surface; Therapeutic; Tissues; Training; Vascular Endothelium; Vascular Permeabilities; Western Blotting; Work; Zinc; acid sphingomyelinase; career; clinically relevant; improved; inhibitor/antagonist; insight; intercellular cell adhesion molecule; interest; nanocarrier; novel strategies; overexpression; passive transport; prevent; public health relevance; receptor; transcytosis

 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.

 描述(申请人提供)：血管内部的内皮细胞阻碍药物载体从血流到病变组织的运输。为了克服这一障碍，药物载体可以针对细胞表面受体，介导跨细胞穿越这一障碍。然而，药物载体在转运后脱离屏障并通过基本的细胞外基质(ECM)迁移的机制仍不清楚。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，这项工作将指导转译药物输送，用于治疗需要跨内皮细胞转运的疾病。作为一个临床相关的应用实例，这项提案将在针对细胞间黏附分子-1(ICAM-1)并携带酶货物(酸性鞘磷脂酶，ASM)的聚合物纳米载体的情况下探索这些问题，用于治疗A-B型尼曼-皮克病，这是一种溶酶体储存障碍，由溶酶体ASM缺乏引起，导致严重的肺和脑功能障碍。具体地说，ICAM-1是研究药物载体转运机制的一个有趣和相关的靶点，因为它在病理条件下在内皮细胞上高度表达，并促进天然(白细胞)和人工(ICAM-1靶向药物载体)配体跨越内皮屏障的转运。初步结果表明，ICAM-1与基质金属蛋白酶(MMPs)，特别是基质金属蛋白酶(MMPs)，尤其是MMP9和MMP2之间存在相互作用。初步结果表明，载体与ICAM-1结合可引起内皮细胞分泌MMP9和MMP2，而MMP9参与裂解ICAM-1。因此，假设载体与ICAM-1结合时释放的基质金属蛋白酶导致(A)ICAM-1裂解，允许载体在跨细胞后从屏障上分离，以及(B)ECM降解，允许载体穿透组织。为了研究这些假说，本研究在细胞培养和动物模型中研究了基质金属蛋白酶-9和基质金属蛋白酶-2在(1)ICAM-1的裂解，(2)载体通过内皮屏障的运输，以及(3)细胞外基质降解对载体穿透的贡献。首先，采用ELISA法和Western blotting法检测基质金属蛋白酶的分泌和细胞间黏附分子-1的裂解，以及基质金属蛋白酶介导的细胞间黏附分子-1的释放与载体生物分布的相关性。第二，放射性同位素示踪将被用来检查载体通过ICAM-1裂解穿过内皮屏障的情况。第三，将使用明胶酶谱、荧光显微镜、免疫组织化学和透射电子显微镜成像来评估ECM的降解和载体渗透。在每种情况下，细胞培养和缺乏基质金属蛋白酶-9和/或基质金属蛋白酶-2的动物模型将被用来确定它们在这些过程中的作用。通过完成这些目标，这项工作将改进一些需要跨越不透内皮屏障的药物治疗。这项研究也将对 将PI培训为独立的研究人员，从而使未来的研究能够促进生物医学技术的进步。