2 research outputs found
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 polyÂ(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
polyÂ(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