title: Polyplex-induced cytosolic nuclease activation leads to differential transgene expression.
abstract: Cytosolic nucleases have been proposed to play an important role in limiting the effectiveness of polyplex-based gene delivery agents. In order to explore the effect of cell membrane disruption on nuclease activation, nuclease activity upon polyplex uptake and localization, and nuclease activity upon gene expression, we employed an oligonucleotide molecular beacon (MB). The MB was incorporated as an integral part of the polymer/DNA polyplex, and two-color flow cytometry experiments were performed to explore the relationship of MB cleavage with propidium iodide (PI) uptake, protein expression, and polyplex uptake. In addition, confocal fluorescence microcopy was performed to examine both polyplex and cleaved MB localization. The impact of cell membrane disruption was also probed using whole-cell patch clamp measurement of the plasma membrane's electrical conductance. Differential activation of cytosolic nuclease was observed with substantial activity for B-PEI and G5 PAMAM dendrimer (G5), less cleavage for jetPEI, and little activity for L-PEI. jetPEI and L-PEI exhibited substantially greater transgene expression, consistent with the lower amounts of MB oligonucleotide cleavage observed. Cytosolic nuclease activity, although dependent on the choice of polymer employed, was not related to the degree of cell plasma membrane disruption that occurred as measured by PI uptake or whole-cell patch clamp.
authors.affiliation: Department of Biomedical Engineering, Michigan Nanotechnology Institute for Medicine and Biological Sciences, University of Michigan , Ann Arbor, Michigan 48019, United States.
authors.forename: Rahul
authors.initials: R
authors.lastname: Rattan
authors.forename: Sriram
authors.initials: S
authors.lastname: Vaidyanathan
authors.forename: Gordon S-H
authors.initials: GS
authors.lastname: Wu
authors.forename: Anisha
authors.initials: A
authors.lastname: Shakya
authors.forename: Bradford G
authors.initials: BG
authors.lastname: Orr
authors.forename: Mark M
authors.initials: MM
authors.lastname: Banaszak Holl
mesh_terms: D050091:Dendrimers / Q000737:chemistry; D003851:Deoxyribonucleases / Q000378:metabolism*; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D018408:Patch-Clamp Techniques; D016062:Porosity; D011419:Propidium / Q000378:metabolism; D014162:Transfection; D019076:Transgenes / Q000235:genetics*
chemical_list: D050091:Dendrimers; C104700:PAMAM Starburst; D011419:Propidium; D003851:Deoxyribonucleases