Development of a borreliosis assay: Mannan coated polyethylene sinter bodies as a new platform technology.

Rapid diagnosis of Lyme borreliosis has been carried out on chemically modified porous polyethylene sinter bodies. Photografting of 2-propenol on sinter body's surface was performed as a first step, introducing active hydroxyl groups as a result of polyalcohol formation. The hydroxyl groups were used for further immobilization and could be linked via 3-aminopropyltriethoxysilane (APTES) to polysaccharides like mannan. Prone to coupling, mannan was activated using N, N'-disuccinimidyl carbonate (DSC) to allow smooth reaction with the primary amine groups of the silane layer. In a final preparation step, a recombinant fusion protein consisting of the mannan-binding domain of the lectin Concanavalin A (ConA) and a specific Borrelia surface antigen was immobilized by self-organization on the mannan surface. The fusion protein was used as biological interface structure. This strategy is highly efficient and resulted in a defined orientation of the antigen part of the fusion protein. Rapid and convenient differentiation could be then established between Borrelia-negative and a -positive serum even in 1000-fold diluted samples and detection of Lyme borreliosis in a rather early stage is likely. Furthermore, this generic strategy can be easily transferred to other bacterial or viral antigen structures.

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.

Thrombin-Inhibiting Anticoagulant Liposomes: Development and Characterization.

Many peptides and peptidomimetic drugs suffer from rapid clearance in vivo; this can be reduced by increasing their size through oligomerization or covalent conjugation with polymers. As proof of principle, an alternative strategy for drug oligomerization is described, in which peptidomimetic thrombin inhibitors are incorporated into the liposome surface. For this purpose, the inhibitor moieties were covalently coupled to a palmitic acid residue through a short bifunctionalized ethylene glycol spacer. These molecules were directly added to the lipid mixture used for liposome preparation. The obtained liposomes possess strong thrombin inhibitory potency in enzyme kinetic measurements and anticoagulant activity in plasma. Their strong potency and positive ζ potential indicate that large amounts of the benzamidine-derived inhibitors are located on the surface of the liposomes. This concept should be applicable to other drug molecules that suffer from rapid elimination and allow covalent modification with a suitable fatty acid residue.