title: Phosphonium-containing polyelectrolytes for nonviral gene delivery.
abstract: Nonviral gene therapy focuses intensely on nitrogen-containing macromolecules and lipids to condense and deliver DNA as a therapeutic for genetic human diseases. For the first time, DNA binding and gene transfection experiments compared phosphonium-containing macromolecules with their respective ammonium analogs. Conventional free radical polymerization of quaternized 4-vinylbenzyl chloride monomers afforded phosphonium- and ammonium-containing homopolymers for gene transfection experiments of HeLa cells. Aqueous size exclusion chromatography confirmed similar absolute molecular weights for all polyelectrolytes. DNA gel shift assays and luciferase expression assays revealed phosphonium-containing polymers bound DNA at lower charge ratios and displayed improved luciferase expression relative to the ammonium analogs. The triethyl-based vectors for both cations failed to transfect HeLa cells, whereas tributyl-based vectors successfully transfected HeLa cells similar to Superfect demonstrating the influence of the alkyl substituent lengths on the efficacy of the gene delivery vehicle. Cellular uptake of Cy5-labeled DNA highlighted successful cellular uptake of triethyl-based polyplexes, showing that intracellular mechanisms presumably prevented luciferase expression. Endocytic inhibition studies using genistein, methyl β-cyclodextrin, or amantadine demonstrated the caveolae-mediated pathway as the preferred cellular uptake mechanism for the delivery vehicles examined. Our studies demonstrated that changing the polymeric cation from ammonium to phosphonium enables an unexplored array of synthetic vectors for enhanced DNA binding and transfection that may transform the field of nonviral gene delivery.
authors.affiliation: Department of Chemistry, Virginia Tech, Blacksburg, Virginia 24061, United States.
authors.forename: Sean T
authors.initials: ST
authors.lastname: Hemp
authors.forename: Michael H
authors.initials: MH
authors.lastname: Allen
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Green
authors.forename: Timothy E
authors.initials: TE
authors.lastname: Long
mesh_terms: D000547:Amantadine / Q000494:pharmacology; D016588:Anticarcinogenic Agents / Q000494:pharmacology; D021941:Caveolae / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000494:pharmacology; D015259:Dopamine Agents / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000737:chemistry / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D009943:Organophosphorus Compounds / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry / Q000494:pharmacology; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: D016588:Anticarcinogenic Agents; D015259:Dopamine Agents; D009943:Organophosphorus Compounds; D011108:Polymers; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C113117:tributylmethyl phosphonium chloride; D004247:DNA; D000547:Amantadine; D019833:Genistein