title: Hydrolytic charge-reversal of PEGylated polyplexes enhances intracellular un-packaging and activity of siRNA.
abstract: Hydrolytically degrading nano-polyplexes (HDG-NPs) that reverse charge through conversion of tertiary amines to carboxylic acids were investigated to improve intracellular un-packaging of siRNA and target gene silencing compared to a non-degradable analog (non-HDG-NPs). Both NP types comprised reversible addition-fragmentation chain-transfer (RAFT) synthesized diblock copolymers of a poly(ethylene glycol) (PEG) corona-forming block and a cationic block for nucleic acid packaging that incorporated butyl methacrylate (BMA) and either dimethylaminoethyl methacrylate (DMAEMA, non-HDG-NPs) or dimethylaminoethyl acrylate (DMAEA, HDG-NPs). HDG-NPs decreased significantly in size and released significantly more siRNA (∼40%) than non-HDG-NPs after 24 h in aqueous solution. While both HDG-NPs and non-HDG-NPs had comparable uptake and cytotoxicity up to 150 nM siRNA doses, HDG-NPs achieved significantly higher target gene silencing of the model gene luciferase in vitro. High resolution FRET confocal microscopy was used to monitor the intracellular un-packaging of siRNA. Non-HDG-NPs had significantly higher FRET efficiency than HDG-NPs, indicating that siRNA delivered from HDG-NPs was more fully un-packaged and therefore had improved intracellular bioavailability.
authors.affiliation: Department of Biomedical Engineering, Vanderbilt Institute for Nanoscale Science and Engineering, Vanderbilt University School of Engineering, Nashville, Tennessee, 37232.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Werfel
authors.affiliation: Department of Biomedical Engineering, Vanderbilt Institute for Nanoscale Science and Engineering, Vanderbilt University School of Engineering, Nashville, Tennessee, 37232.
authors.forename: Corban
authors.initials: C
authors.lastname: Swain
authors.affiliation: Department of Biomedical Engineering, Vanderbilt Institute for Nanoscale Science and Engineering, Vanderbilt University School of Engineering, Nashville, Tennessee, 37232.
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Nelson
authors.affiliation: Department of Biomedical Engineering, Vanderbilt Institute for Nanoscale Science and Engineering, Vanderbilt University School of Engineering, Nashville, Tennessee, 37232.
authors.forename: Kameron V
authors.initials: KV
authors.lastname: Kilchrist
authors.affiliation: Department of Biomedical Engineering, Vanderbilt Institute for Nanoscale Science and Engineering, Vanderbilt University School of Engineering, Nashville, Tennessee, 37232.
authors.forename: Brian C
authors.initials: BC
authors.lastname: Evans
authors.affiliation: Department of Biomedical Engineering, Vanderbilt Institute for Nanoscale Science and Engineering, Vanderbilt University School of Engineering, Nashville, Tennessee, 37232.
authors.forename: Martina
authors.initials: M
authors.lastname: Miteva
authors.affiliation: Department of Biomedical Engineering, Vanderbilt Institute for Nanoscale Science and Engineering, Vanderbilt University School of Engineering, Nashville, Tennessee, 37232.
authors.forename: Craig L
authors.initials: CL
authors.lastname: Duvall
mesh_terms: D000588:Amines / Q000737:chemistry; D002264:Carboxylic Acids / Q000737:chemistry; D045744:Cell Line, Tumor; D002470:Cell Survival; D031541:Fluorescence Resonance Energy Transfer; D006461:Hemolysis; D006801:Humans; D006868:Hydrolysis; D008689:Methacrylates / Q000737:chemistry*; D018613:Microscopy, Confocal; D049329:Nanostructures / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000008:administration & dosage*
chemical_list: D000588:Amines; D002264:Carboxylic Acids; D008689:Methacrylates; D034741:RNA, Small Interfering; D011092:Polyethylene Glycols; C049840:2-(dimethylamino)ethyl methacrylate; C016284:butyl methacrylate
keywords: FRET microscopy; RAFT; charge-reversal; hydrolytically degrading; polyplexes; siRNA