[Show abstract][Hide abstract] ABSTRACT: Numerous applications of conventional and biogenic magnetic nanoparticles (MNPs), such as in diagnostics, immunomagnetic separations, and magnetic cell labeling, require the immobilization of antibodies. This is usually accomplished by chemical conjugation, which, however, has several disadvantages, such as poor efficiency and the need for coupling chemistry. Here, we describe a novel strategy to display a functional camelid antibody fragment (nanobody) from an alpaca (Lama pacos) on the surface of bacterial biogenic magnetic nanoparticles (magnetosomes). Magnetosome-specific expression of a red fluorescent protein (RFP)-binding nanobody (RBP) in vivo was accomplished by genetic fusion of RBP to the magnetosome protein MamC in the magnetite-synthesizing bacterium Magnetospirillum gryphiswaldense. We demonstrate that isolated magnetosomes expressing MamC-RBP efficiently recognize and bind their antigen in vitro and can be used for immunoprecipitation of RFP-tagged proteins and their interaction partners from cell extracts. In addition, we show that coexpression of monomeric RFP (mRFP or its variant mCherry) and MamC-RBP results in intracellular recognition and magnetosome recruitment of RFP within living bacteria. The intracellular expression of a functional nanobody targeted to a specific bacterial compartment opens new possibilities for in vivo synthesis of MNP-immobilized nanobodies. Moreover, intracellular nanotraps can be generated to manipulate bacterial structures in live cells.
Applied and Environmental Microbiology 09/2011; 77(17):6165-71. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The rapid and ongoing discovery of new disease related biomarkers leads to a dramatic paradigm change in human healthcare and constitutes the basis for a truly personalized medicine. Molecular imaging enables early detection and classification of human diseases and provides valuable data for optimized, target-oriented therapies. By now, the biochemical and physiological properties of antibody derivatives or alternative protein scaffolds can be engineered for the detection of a wide range of target structures. The successful application of these reagents in animals, xenograft models and cells in preclinical research clearly demonstrate their utility for molecular imaging. Despite these promising perspectives, only a few antibodies and recombinant proteins are used yet for molecular imaging in human medicine. Especially the high safety demands and the need to eliminate off target effects in humans require extensive research and development efforts.
Current opinion in biotechnology 06/2011; 22(6):882-7. · 7.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Molecular conjugates comprising targeting ligands hold great promise for site-specific gene delivery to distant tumors and individual organs including the lung. Here we show that prostaglandin I2 analogues can be used to improve gene transfer efficiency of polyethylenimine (PEI) gene vectors on bronchial and alveolar epithelial cells in vitro and lungs of mice in vivo. Prostacyclin (IP1) receptor expression was confirmed in pulmonary epithelial cell lines by western blot. Iloprost (ILO) and treprostinil (TRP), two prostaglandin I2 analogues, were conjugated to fluorescein-labeled BSA (FLUO-BSA) and compared for IP1 receptor binding/uptake in different lung cell lines. Binding of FLUO-BSA-ILO was 2-4-fold higher than for FLUO-BSA-TRP and could be specifically inhibited by free ILO and IP1 receptor antagonist CAY10449. Internalization of FLUO-BSA-ILO was confirmed by confocal microscopy. Molecular conjugates of PEI and ILO (PEI-g-ILO) were synthesized with increasing coupling degree (F(ILO) (ILO:PEI) = 2, 5, 8, 16) and analyzed for DNA binding, particle formation and transfection efficiency. At optimized conditions (N/P 4, F(ILO) = 5), gene expression using PEI-g-ILO was significantly up to 46-fold higher than for PEI gene vectors and specifically inhibited by CAY10449. Gene expression in the lungs of mice after aerosol delivery was 14-fold higher with PEI-g-ILO F(ILO) = 5 than for PEI. We suggest that targeting of IP1 receptor using ILO represents a promising approach to improve pulmonary gene transfer.