Recent advances in magnetofection and its potential to deliver siRNAs in vitro.
This chapter describes how to design and conduct experiments to deliver siRNA to adherent mammalian cells 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. These magnetic complexes are targeted to the cell surface by the application of a magnetic gradient field. In this chapter, first we describe the synthesis of magnetic nanoparticles for magnetofection and the association of siRNA with the magnetic components of the transfection complex. Second, a simple protocol is described in order to evaluate magnetic responsiveness of the magnetic siRNA transfection complexes and estimate the complex loading with magnetic nanoparticles. Third, protocols are provided for the preparation of magnetic lipoplexes and polyplexes of siRNA, magnetofection, downregulation of gene expression, and the determination of cell viability. The addition of INF-7 peptide, a fusogenic peptide, to the magnetic transfection triplexes improved gene silencing in HeLa cells. The described protocols are also valuable for screening vector compositions and novel magnetic nanoparticle preparations to optimize siRNA transfection by magnetofection in every cell type.
Mykhaylyk
Olga
O
Institute of Experimental Oncology, Technische Universität München, Munich, Germany.
Zelphati
Olivier
O
Hammerschmid
Edelburga
E
Anton
Martina
M
Rosenecker
Joseph
J
Plank
Christian
C
D002291:Carcinoma, Papillary / Q000378:metabolism / Q000628:therapy; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000008:administration & dosage / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008175:Lung Neoplasms / Q000378:metabolism / Q000628:therapy; D008280:Magnetics; D053758:Nanoparticles / Q000737:chemistry; D010455:Peptides / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000235:genetics; D014162:Transfection / Q000379:methods*
C526981:INF7 peptide; D010455:Peptides; D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins