A novel method for the assessment of targeted PEI-based nanoparticle binding based on a static surface plasmon resonance system.
A novel method for the assessment of targeted PEI-based nanoparticle binding based on a static surface plasmon resonance system.
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DOI：
10.1021/ac402001q
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发表时间：
2014-07
影响因子：
7.4
通讯作者：
Sabrina Höbel;D. Vornicescu;Marius Bauer;D. Fischer;M. Keusgen;A. Aigner
中科院分区：
文献类型：
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作者：
Sabrina Höbel;D. Vornicescu;Marius Bauer;D. Fischer;M. Keusgen;A. Aigner
核酸的传递是基因治疗或治疗性基因敲除的主要障碍，因此，开发智能和安全的纳米颗粒作为载体系统正在进行深入的研究。引入配体进行靶向递送是人们主要感兴趣的事情。在这里，我们描述了一种新的方法来分析抗体功能化纳米颗粒的结合特性，使用静态试管系统中的表面等离子体共振(SPR)。通过将表皮生长因子受体(EGFR)特异性抗体西妥昔单抗通过聚乙二醇亚胺(PEI)与聚乙二醇亚胺(PEI)进行化学偶联，以及随后的DNA或siRNA络合，我们生成了具有低表面电荷的靶向纳米网络。观察到抗体介导的摄取EGFR过表达的细胞。SPR测量使用一种新型的基于蛋白A的夹心系统将目标受体固定在其正确的空间取向上，从而可以分析与EGFR结合的特定PEI-PEG-西妥昔单抗，并确定结合亲和力。重要的是，我们的基于试管的SPR分析系统也适用于监测配体介导的纳米颗粒结合，而不需要对流或剪应力。我们得出的结论是，我们的SPR夹心系统允许实时准确地分析配体功能化纳米颗粒的结合，因此我们建立了SPR用于体外评估配体修饰以产生靶向纳米颗粒。
The delivery of nucleic acids is a major hurdle in gene therapy or therapeutic gene knockdown, and the development of intelligent and safe nanoparticles as carrier systems is thus under intense investigation. The introduction of ligands for their targeted delivery is of major interest. Here, we describe a novel approach for the analysis of the binding properties of antibody-functionalized nanoparticles, using surface plasmon resonance (SPR) in a static cuvette system. By chemical coupling of the Epidermal Growth Factor Receptor (EGFR)-specific antibody cetuximab to poly(ethylene imine) (PEI) via a PEG-spacer and subsequent DNA or siRNA complexation, we generated targeted nanoplexes with low surface charge. Antibody-mediated uptake into EGFR overexpressing cells was observed. SPR measurements with use of a novel, protein A-based sandwich system for the immobilization of the target receptor in its correct steric orientation allowed the analysis of the specific PEI-PEG-cetuximab binding to EGFR and the determination of binding affinities. Importantly, our cuvette-based SPR assay system was also suitable for the monitoring of ligand-mediated nanoparticle binding, without convection or shear stress. We conclude that our SPR sandwich system allows the precise analysis of the binding of ligand-functionalized nanoparticles in real-time, and we thus establish SPR for the in vitro evaluation of ligand modifications for generating targeted nanoparticles.