Establishment of magnetofection - a novel method using superparamagnetic nanoparticles and magnetic force to enhance and to target nucleic acid delivery

摘要

Among the physical methods of drug localization, especially magnetic drug targeting promises great potential. In this method, an anti-cancer drug is bound to magnetic particles and an external magnetic field can guide the administered magnetic particle-drug complex to the desired site. The objective of this thesis was to apply the principle of magnetic drug targeting to the delivery of nucleic acids in cell culture and in vivo. To establish the method of magnetic nucleic acid targeting (magnetofection), the characteristics (sizes and organization) of different superparamagnetic iron oxide nanoparticles coated with cationic or anionic polymers (termed “trMAGs”, synthesized by Chemicell GmbH Berlin), the binding of DNA to the magnetic beads, transfections with the different types of magnetic particles, the mechanism of magnetofection, optimization of magnetofection, the gene transfer efficiency of magnetofections compared to standard (conventional) transfections, magnetic field-guided localization of gene transfer, magnetofection of a variety of cells and the applicability of magnetofection in vivo, were examined. In binding studies, it turned out that efficient binding of charged DNA vectors to charged magnetic particles could be achieved by salt-induced colloid aggregation. Incubation of cells with magnetic particle/DNA associates (magnetofectins) resulted in gene transfer and application of a magnetic field significantly increased gene expression. Additionally, polyethylenimine (PEI) had an enhancing effect on magnetofection efficiency. Mechanistic studies revealed that the paramagnetic vectors are concentrated efficiently by magnetic force on the cell surface within minutes and the predominant uptake mechanism is endocytosis. Comparison of magnetofections and the corresponding standard transfections (same vectors but without magnetic particles and no applied magnetic field) showed that with magnetofection the gene transfer efficiency was usually significantly enhanced (up to 971-fold), the nucleic acid dose-response profile could be improved and the incubation times (of cells with vectors) could be reduced from hours to minutes. Finally, in animal experiments (injection into ear veins of pigs, into ear arteries of rabbits and into ilea of rats) it was demonstrated that magnetofection enables targeted and efficient gene transfer in vivo.