Targeting of perfluorocarbon nanoemulsions for imaging of specific cell surface epitopes by non-invasive in-vivo 19F MRI

通过非侵入性体内 19F MRI 靶向全氟化碳纳米乳剂对特定细胞表面表位进行成像

• 批准号: 170888146
• 负责人: Professor Dr. Jürgen Schrader
• 金额: --
• 依托单位: Institut für Molekulare Medizin I
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2010
• 资助国家: 德国
• 起止时间: 2009-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/170888146?language=en
• 关键词: Targeting; perfluorocarbon; nanoemulsions; imaging; specific

In the current funding period, we successfully demonstrated that 19F MRI (magnetic resonance imaging) in combination with perfluorocarbon nanoemulsions (PFC-NE) is a sensitive diagnostic tool for inflammation imaging in a variety of disease models. Based on this prior work, we now plan to substantially extend the application field of this approach for the specific in-vivo targeting of individual cell surface epitopes. We will use a sterol-based post-insertion technique (SPIT) to modify PFC-NE with specific antibodies, Fab-fragments or single-chain antibodies. In case of SPIT, a ligand is coupled to a sterol-PEG-anchor (PEG = polyethylenglycol), which contains a reactive group at its membrane-distal end. Subsequently, this construct is inserted into the lipid layer of preformed PFC-NE. SPIT enables the coupling of ligands to PFC-NE which would be otherwise destroyed during the high-pressure homogenization process. We already exploited this strategy successfully for the visualization of developing thrombi by coupling alpha-2-antiplasmin peptide to the surface of PEGylated PFC-NE.Using this technique we will experimentally address two important biological questions: 1) Selective visualization of preexisting venous thrombi To visualize existing thrombi, we will modify PFC-NE with IgG and Fab-fragments of JON/A(PE) and with a single-chain-antibody (LB24) which both bind to the activated form of the GPIIb/IIIa receptor on murine platelets. All antibodies are well characterized and will be provided by Professor Nieswandt (Würzburg) and Professor Peter (Melbourne, Australia). After coupling to PFC-NE, the sensitivity and specificity of thrombus detection will be verified in-vivo with 19F MRI using an established thrombosis model. 2) Tracking of epicardial mesenchymal progenitor cells (EPDCs) and monocytes We have recently discovered that EPDCs, which develop after myocardial infarction and are able to differentiate into smooth muscle cells and cardiomyocytes, are capable to endocytose PFC-NE in-vivo. Therefore, EPDCs as well as monocytes can be visualized by 19F MRI after intravenous injection. To selectively label and track EPDCs or monocytes, we will develop target-specific PFC-NE. To this end, cell surface proteins which are exclusively expressed by EPDCs or monocytes will be identified by mass spectrometry (Cooperation Professor Stühler, Düsseldorf). Subsequently, monoclonal antibodies are raised against these epitopes (Cooperation Professor Nieswandt) and coupled to PFC-NE by the SPIT-technique for selective non-invasive 19F MRI of EPDCs/monocytes after myocardial infarction.The overall goal of this project is to develop a versatile platform for the in-vivo visualization of specific target-structures by 19F MRI which can be applied to biologically relevant questions.

在当前的资助期间，我们成功地证明了19F MRI(磁共振成像)与全氟碳纳米乳剂(PFC-NE)相结合是一种在各种疾病模型中进行炎症成像的敏感诊断工具。在之前工作的基础上，我们现在计划大幅扩展这种方法的应用领域，用于在体内针对单个细胞表面表位的特定靶向。我们将使用基于固醇的插入后技术(SPIT)来用特定的抗体、FAB片段或单链抗体来修饰PFC-NE。在SPIT的情况下，配体偶联到甾醇-聚乙二醇锚(PEG=聚乙二醇)上，该锚在其膜的远端含有反应基团。随后，该构建体被插入到预制的PFC-NE的脂层中。SPIT使配体能够与PFC-NE偶联，否则在高压均质过程中会被破坏。我们已经将这一策略成功地应用于通过将α-2-抗纤溶酶多肽偶联到聚乙二醇化的PFC-NE表面来显示血栓的发展。利用这一技术，我们将实验地解决两个重要的生物学问题：1)选择性地显示先前存在的静脉血栓为了显示现有的血栓，我们将用Jon/A的Ig G和Fab片段(PE)和与小鼠血小板上的GPIIb/IIIa受体的激活形式结合的单链抗体(Lb24)来修饰PFC-NE。所有抗体都具有良好的特性，将由Nieswandt教授(维尔茨堡)和Peter教授(澳大利亚墨尔本)提供。在与PFC-NE偶联后，血栓检测的敏感性和特异性将使用已建立的血栓形成模型通过19F MRI在体内进行验证。2)心外膜间充质前体细胞(EPDCs)和单核细胞的示踪我们最近发现，心肌梗死后发育的EPDCs能够在体内内吞PFC-NE，并能分化为平滑肌细胞和心肌细胞。因此，静脉注射后的19F磁共振成像可以显示EPDCs和单核细胞。为了选择性地标记和跟踪EPDCs或单核细胞，我们将开发靶向特异性PFC-NE。为此，EPDCs或单核细胞独有表达的细胞表面蛋白将通过质谱学进行鉴定(合作教授施特勒，杜塞尔多夫)。随后，针对这些表位提出了单抗(合作教授Nieswandt)，并通过SPIT技术将其偶联到PFC-NE上，用于心肌梗死后EPDCs/单核细胞的选择性非侵入性19F MRI。本项目的总体目标是开发一个通用的19F MRI平台，用于体内通过19F MRI显示特定的靶结构，可用于生物学相关问题。