A Novel Method for the Assessment of Targeted PEI-Based Nanoparticle Binding Based on a Static Surface Plasmon Resonance System

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

Polyelectrolyte Complexes of DNA and Linear PEI: Formation, Composition and Properties

In the present study, the complexation between linear 13.4 kDa poly­(ethylene imine) (LPEI) and plasmid DNA was investigated. Analytical ultracentrifugation (AUC) was used for size and molar mass determination. Additionally, the morphology was studied by scanning force microscopy. The polyplex formation was investigated in a wide range of PEI nitrogen to DNA phosphate ratios (N/P). At N/P ratios below 1, the PEI/DNA complex formation is characterized by an incomplete DNA condensation and the formation of the primary DNA/PEI complexes. The merging of the initially formed polyplexes occurs at N/P ∼2, resulting in the formation of polyplexes with much larger size and high aggregation rate. Stable and uniform polyplexes were formed at N/P > 10, with average sizes of the polyplexes of about 170 ± 65 nm. The content of uncomplexed PEI chains in the polyplex dispersion was estimated at four different N/P ratios, 6.2, 11.6, 28.6, and 57.8, by combining preparative centrifugation with a copper complex assay and by sedimentation velocity analysis as an alternative method. It is demonstrated that virtually all added PEI binds to the DNA at N/P < 2.5; further addition of PEI results in the appearance of a large amount of free PEI in solution. Nevertheless, PEI is able to bind in the whole range of N/P ratios tested. According to the data collected by sedimentation velocity analysis and scanning force microscopy, the single PEI/DNA complexes are composed on average of 8 to 32 single condensed DNA plasmids and 70 ± 25 PEI molecules