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

在当前的资助期内，我们成功地证明了19 F MRI（磁共振成像）与全氟化碳纳米乳剂（PFC-NE）的结合是一种敏感的诊断工具，用于各种疾病模型中的炎症成像。基于这一先前的工作，我们现在计划实质性地扩展这种方法的应用领域，用于个体细胞表面表位的特异性体内靶向。我们将使用基于甾醇的后插入技术（SPIT）来用特异性抗体、Fab片段或单链抗体修饰PFC-NE。在SPIT的情况下，配体与甾醇-PEG-锚（PEG =聚乙二醇）偶联，其在其膜远端含有反应性基团。随后，将该构建体插入预先形成的PFC-NE的脂质层中。SPIT能够将配体偶联到PFC-NE，否则PFC-NE将在高压均质化过程中被破坏。我们已经成功地利用这种策略，通过将α-2-抗纤溶酶肽偶联到聚乙二醇化的PFC-NE表面来可视化血栓的形成。使用这种技术，我们将实验性地解决两个重要的生物学问题：1）预先存在的静脉血栓的选择性可视化为了可视化现有的血栓，我们将用IgG和JON/A（PE）的Fab片段以及用单链抗体（LB 24）修饰PFC-NE，所述抗体都与鼠血小板上GPIIb/IIIa受体的活化形式结合。所有抗体均经过充分表征，并将由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，将使用已建立的血栓形成模型用19 F MRI在体内验证血栓检测的灵敏度和特异性。2)心外膜间充质祖细胞（EPDC）和单核细胞的追踪我们最近发现，心肌梗死后发育并能够分化为平滑肌细胞和心肌细胞的EPDC能够在体内内吞PFC-NE。因此，静脉注射后，EPDC以及单核细胞可以通过19 F MRI可视化。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，将通过质谱法（Cooperation Professor Stühler，Düsseldorf）鉴定仅由EPDC或单核细胞表达的细胞表面蛋白。随后，针对这些表位产生单克隆抗体（合作教授Nieswandt），并通过SPIT技术偶联到PFC-NE，用于心肌梗死后EPDCs/单核细胞的选择性非侵入性19 F MRI。本项目的总体目标是开发一个通用平台，用于通过19 F MRI对特定靶结构进行体内可视化，该平台可应用于生物学相关问题。

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.