title: Magnetic nanoparticle and magnetic field assisted siRNA delivery in vitro.
abstract: This chapter describes how to design and conduct experiments to deliver siRNA to adherent cell cultures in vitro by magnetic force-assisted transfection using self-assembled complexes of small interfering RNA (siRNA) and cationic lipids or polymers that are associated with magnetic nanoparticles (MNPs). These magnetic complexes are targeted to the cell surface by the application of a gradient magnetic field. A further development of the magnetic drug-targeting concept is combining it with an ultrasound-triggered delivery using magnetic microbubbles as a carrier for gene or drug delivery. For this purpose, selected MNPs, phospholipids, and siRNAs are assembled in the presence of perfluorocarbon gas into flexible formulations of magnetic lipospheres (microbubbles). Methods are described how to accomplish the synthesis of magnetic nanoparticles for magnetofection and how to test the association of siRNA with the magnetic components of the transfection vector. A simple method is described to evaluate magnetic responsiveness of the magnetic siRNA transfection complexes and estimate the complex loading with magnetic nanoparticles. Procedures are provided for the preparation of magnetic lipoplexes and polyplexes of siRNA as well as magnetic microbubbles for magnetofection and downregulation of the target gene expression analysis with account for the toxicity determined using an MTT-based respiration activity test. A modification of the magnetic transfection triplexes with INF-7, fusogenic peptide, is described resulting in reporter gene silencing improvement in HeLa, Caco-2, and ARPE-19 cells. The methods described can also be useful for screening vector compositions and novel magnetic nanoparticle preparations for optimized siRNA transfection by magnetofection in any cell type.
authors.affiliation: Institute of Experimental Oncology, Klinikum rechts der Isar der Technischen Universität München, Ismaninger Strasse 22, Munich, 81675, Germany, olga.mykhaylyz@lrz.tu-muenchen.de.
authors.forename: Olga
authors.initials: O
authors.lastname: Mykhaylyk
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Sanchez-Antequera
authors.forename: Dialechti
authors.initials: D
authors.lastname: Vlaskou
authors.forename: Maria Belen
authors.initials: MB
authors.lastname: Cerda
authors.forename: Mehrdad
authors.initials: M
authors.lastname: Bokharaei
authors.forename: Edelburga
authors.initials: E
authors.lastname: Hammerschmid
authors.forename: Martina
authors.initials: M
authors.lastname: Anton
authors.forename: Christian
authors.initials: C
authors.lastname: Plank
mesh_terms: D018938:Caco-2 Cells; D045744:Cell Line, Tumor; D019069:Cell Respiration; D004337:Drug Carriers / Q000737:chemistry*; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005466:Fluorocarbons / Q000737:chemistry; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D007097:Imines / Q000737:chemistry; D007457:Iodine Radioisotopes; D060526:Magnetic Fields; D058185:Magnetite Nanoparticles / Q000737:chemistry*; D045423:Microbubbles; D010743:Phospholipids / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011095:Polyethylenes / Q000737:chemistry; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D014465:Ultrasonics
chemical_list: D004337:Drug Carriers; D005466:Fluorocarbons; D007097:Imines; D007457:Iodine Radioisotopes; D058185:Magnetite Nanoparticles; D010743:Phospholipids; D011095:Polyethylenes; D034741:RNA, Small Interfering; C505405:poly(ethylene imine)