title: Polycation-induced cell membrane permeability does not enhance cellular uptake or expression efficiency of delivered DNA.
abstract: Polycationic materials commonly used to delivery DNA to cells are known to induce cell membrane porosity in a charge-density dependent manner. It has been suggested that these pores may provide a mode of entry of the polymer-DNA complexes (polyplexes) into cells. To examine the correlation between membrane permeability and biological activity, we used two-color flow cytometry on two mammalian cell lines to simultaneously measure gene expression of a plasmid DNA delivered with four common nonviral vectors and cellular uptake of normally excluded fluorescent dye molecules of two different sizes, 668 Da and 2 MDa. We also followed gene expression in cells sorted based on the retention of endogenous fluorescein. We have found that cell membrane porosity caused by polycationic vectors does not enhance internalization or gene expression. Based on this single-cell study, membrane permeability is found to be an unwanted side effect that limits transfection efficiency, possibly through leakage of the delivered nucleic acid through the pores prior to transcription and translation and/or activation of cell defense mechanisms that restrict transgene expression.
authors.affiliation: Department of Chemistry, Macromolecular Science and Engineering Program, and Michigan Nanotechnology Institute for Medicine and Biological Sciences (MNIMBS), University of Michigan, 911 North University Avenue, Ann Arbor, MI 48109-1055, USA.
authors.forename: Lisa E
authors.initials: LE
authors.lastname: Prevette
authors.forename: Douglas G
authors.initials: DG
authors.lastname: Mullen
authors.forename: Mark M Banaszak
authors.initials: MM
authors.lastname: Holl
mesh_terms: D000818:Animals; D019556:COS Cells; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002522:Chlorocebus aethiops; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D050091:Dendrimers / Q000009:adverse effects / Q000737:chemistry; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000009:adverse effects / Q000737:chemistry; D006801:Humans; D008962:Models, Theoretical; D011073:Polyamines / Q000494:pharmacology*; D000071228:Polyelectrolytes; D014162:Transfection
chemical_list: D004247:DNA; D050091:Dendrimers; D011073:Polyamines; D000071228:Polyelectrolytes; C104700:PAMAM Starburst