title: Screening nylon-3 polymers, a new class of cationic amphiphiles, for siRNA delivery.
abstract: Amphiphilic nucleic acid carriers have attracted strong interest. Three groups of nylon-3 copolymers (poly-β-peptides) possessing different cationic/hydrophobic content were evaluated as siRNA delivery agents in this study. Their ability to condense siRNA was determined in SYBR Gold assays. Their cytotoxicity was tested by MTT assays, their efficiency of delivering Alexa Fluor-488-labeled siRNA intracellularly in the presence and absence of uptake inhibitors was assessed by flow cytometry, and their transfection efficacies were studied by luciferase knockdown in a cell line stably expressing luciferase (H1299/Luc). Endosomal release was determined by confocal laser scanning microscopy and colocalization with lysotracker. All polymers efficiently condensed siRNA at nitrogen-to-phosphate (N/P) ratios of 5 or lower, as reflected in hydrodynamic diameters smaller than that at N/P 1. Although several formulations had negative zeta potentials at N/P 1, G2C and G2D polyplexes yielded >80% uptake in H1299/Luc cells, as determined by flow cytometry. Luciferase knockdown (20-65%) was observed after transfection with polyplexes made of the high molecular weight polymers that were the most hydrophobic. The ability of nylon-3 polymers to deliver siRNA intracellularly even at negative zeta potential implies that they mediate transport across cell membranes based on their amphiphilicity. The cellular uptake route was determined to strongly depend on the presence of cholesterol in the cell membrane. These polymers are, therefore, very promising for siRNA delivery at reduced surface charge and toxicity. Our study identified nylon-3 formulations at low N/P ratios for effective gene knockdown, indicating that nylon-3 polymers are a new, promising type of gene delivery agent.
authors.affiliation: Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University , Detroit, Michigan 48201, United States.
authors.forename: Venkatareddy
authors.initials: V
authors.lastname: Nadithe
authors.forename: Runhui
authors.initials: R
authors.lastname: Liu
authors.forename: Bryan A
authors.initials: BA
authors.lastname: Killinger
authors.forename: Sara
authors.initials: S
authors.lastname: Movassaghian
authors.forename: Na Hyung
authors.initials: NH
authors.lastname: Kim
authors.forename: Anna B
authors.initials: AB
authors.lastname: Moszczynska
authors.forename: Kristyn S
authors.initials: KS
authors.lastname: Masters
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Gellman
authors.forename: Olivia M
authors.initials: OM
authors.lastname: Merkel
mesh_terms: D002412:Cations / Q000737:chemistry*; D018014:Gene Transfer Techniques; D015394:Molecular Structure; D009757:Nylons / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry*; D034741:RNA, Small Interfering / Q000737:chemistry*; D014162:Transfection / Q000379:methods*
chemical_list: D002412:Cations; D009757:Nylons; D011108:Polymers; D034741:RNA, Small Interfering
keywords: RNAi; nylon-3; polyplexes; siRNA; transfection