AFM visualization of sub-50 nm polyplex disposition to the nuclear pore complex without compromising the integrity of the nuclear envelope

It has been questioned as to whether polyplexes in the cytoplasmcan reach the nuclear compartment and if so in what form. By applying atomic force microscopy (AFM) to the nuclear envelope and the nuclear pore complexes, we demonstrate that disposition of polyethylenimine (PEI)/DNA polyplexes that were microinjected into the oocytes of Xenopus laevis, as an example of a non-dividing cell, is exclusive to the nuclear pore complex (NPC). AFM images show NPCs clogged only with sub-50 nm polyplexes. This mode of disposition neither altered the morphology/ integrity of the nuclearmembrane nor the NPC. AFM images further show polyplexes on the nucleoplasmic side of the envelope, presumably indicating species in transit. Transmission electron microscopy studies of ruptured nuclei from transfected human cell lines demonstrate the presence of sub-50 nm particles resembling polyplexes in morphology compared with control preparations. (C) 2016 Published by Elsevier B.V.