Nuclear delivery of NFκB-assisted DNA/polymer complexes: plasmid DNA quantitation by confocal laser scanning microscopy and evidence of nuclear polyplexes by FRET imaging

Abstract

Quantification of a plasmid DNA (pDNA) and investigation of its polymer-associated state in the nucleus are crucial to evaluate the effectiveness of a gene-delivery system. This study was conducted with p3NF-luc-3NF, a pDNA-bearing optimized κB motif to favour NFκB-driven nuclear import. Here, a quantification of pDNA copies in the nucleus was performed by real-time confocal laser scanning microscopy in HeLa and C2C12 cells transfected with linear polyethylenimine or histidylated polylysine. Förster Resonance Energy Transfer (FRET) from the fluorescein-p3NF-luc-3NF donor to the co-localized rhodamine-polymer acceptor was carried out to investigate whether the pDNA was still condensed with the polymer in the nucleus. Upon 5 h of transfection, the nuclear amount of p3NF-luc3NF was ∼1500 copies in both cell lines whereas that of pTAL-luc, a 3NF-free counterpart pDNA, was less than 250. This quantity of p3NF-luc-3NF dropped dramatically to that of pTAL-luc in the presence of the BAY 11-7085, an inhibitor of NFκB activation. These data strongly support a nuclear import of p3NF-luc3NF mediated by NFκB. Moreover, FRET experiments clearly revealed that most of nuclear pDNA were still condensed with the polymer raising the question of their passage through the nuclear pore complex and their impact on the gene-expression efficiency.

Intracellular partitioning of cell organelles and extraneous nanoparticles during mitosis

“…Also, Pollard et al [62] showed that PEI polyplexes injected in the nucleus were transcriptionally active, questioning the need for cytoplasmic pDNA unloading. In line with these speculations, Breuzard et al [105] found evidence of nuclear polyplexes by FRET imaging. On the contrary, Bieber et al [106] found no evidence of nuclear PEI polyplexes and mainly observed colocalization with lysosomes.…”

Efficient Generation of Mucosal and Systemic Antigen-Specific CD8+T-Cell Responses following Pulmonary DNA Immunization

“…The delayed kinetics of antigen-specific CD8 ϩ T cells observed following pulmonary immunization with PEI-DNA could be a result of the prolonged residence time of the PEI-DNA complexes in the cell nucleus (7,9). Furthermore, prolonged antigen presentation by DCs in the draining lymph nodes of the airways could shape the kinetics of both systemic and locally activated T cells (41).…”

Influence of pDNA availability on transfection efficiency of polyplexes in non-proliferative cells

“…The study was performed with polyplexes made up of linear polyethylenimine (lPEI) or histidinylated lPEI (His 16 lPEI) and of pDNA containing 3NF nuclear localization sequences [25,26]. The investigations were carried out in proliferative, nondividing and differentiated mouse skeletal muscle C2C12 cells.…”