title: Combinatorial optimization of PEG architecture and hydrophobic content improves ternary siRNA polyplex stability, pharmacokinetics, and potency in vivo.
abstract: A rationally-designed library of ternary siRNA polyplexes was developed and screened for gene silencing efficacy in vitro and in vivo with the goal of overcoming both cell-level and systemic delivery barriers. [2-(dimethylamino)ethyl methacrylate] (DMAEMA) was homopolymerized or copolymerized (50mol% each) with butyl methacrylate (BMA) from a reversible addition - fragmentation chain transfer (RAFT) chain transfer agent, with and without pre-conjugation to polyethylene glycol (PEG). Both single block polymers were tested as core-forming units, and both PEGylated, diblock polymers were screened as corona-forming units. Ternary siRNA polyplexes were assembled with varied amounts and ratios of core-forming polymers to PEGylated corona-forming polymers. The impact of polymer composition/ratio, hydrophobe (BMA) placement, and surface PEGylation density was correlated to important outcomes such as polyplex size, stability, pH-dependent membrane disruptive activity, biocompatibility, and gene silencing efficiency. The lead formulation, DB4-PDB12, was optimally PEGylated not only to ensure colloidal stability (no change in size by DLS between 0 and 24h) and neutral surface charge (0.139mV) but also to maintain higher cell uptake (>90% positive cells) than the most densely PEGylated particles. The DB4-PDB12 polyplexes also incorporated BMA in both the polyplex core- and corona-forming polymers, resulting in robust endosomolysis and in vitro siRNA silencing (~85% protein level knockdown) of the model gene luciferase across multiple cell types. Further, the DB4-PDB12 polyplexes exhibited greater stability, increased blood circulation time, reduced renal clearance, increased tumor biodistribution, and greater silencing of luciferase compared to our previously-optimized, binary parent formulation following intravenous (i.v.) delivery. This polyplex library approach enabled concomitant optimization of the composition and ratio of core- and corona-forming polymers (indirectly tuning PEGylation density) and identification of a ternary nanomedicine optimized to overcome important siRNA delivery barriers in vitro and in vivo.
authors.affiliation: Department of Biomedical Engineering, Vanderbilt Institute for Nanoscale Science and Engineering, Vanderbilt University School of Engineering, Nashville, TN, USA.
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, TN, USA.
authors.forename: Meredith A
authors.initials: MA
authors.lastname: Jackson
authors.affiliation: Department of Biomedical Engineering, Vanderbilt Institute for Nanoscale Science and Engineering, Vanderbilt University School of Engineering, Nashville, TN, USA.
authors.forename: Taylor E
authors.initials: TE
authors.lastname: Kavanaugh
authors.affiliation: Department of Biomedical Engineering, Vanderbilt Institute for Nanoscale Science and Engineering, Vanderbilt University School of Engineering, Nashville, TN, USA.
authors.forename: Kellye C
authors.initials: KC
authors.lastname: Kirkbride
authors.affiliation: Department of Biomedical Engineering, Vanderbilt Institute for Nanoscale Science and Engineering, Vanderbilt University School of Engineering, Nashville, TN, USA.
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, TN, USA.
authors.forename: Todd D
authors.initials: TD
authors.lastname: Giorgio
authors.affiliation: Department of Biomedical Engineering, Vanderbilt Institute for Nanoscale Science and Engineering, Vanderbilt University School of Engineering, Nashville, TN, USA. Electronic address: craig.duvall@vanderbilt.edu.
authors.forename: Craig
authors.initials: C
authors.lastname: Duvall
mesh_terms: D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008156:Luciferases / Q000235:genetics; D008689:Methacrylates / Q000737:chemistry*; D051379:Mice; D008819:Mice, Nude; D011092:Polyethylene Glycols / Q000737:chemistry*; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000737:chemistry / Q000493:pharmacokinetics; D014018:Tissue Distribution
chemical_list: D008689:Methacrylates; D034741:RNA, Small Interfering; D011092:Polyethylene Glycols; D008156:Luciferases; C049840:2-(dimethylamino)ethyl methacrylate; C016284:butyl methacrylate
keywords: Cancer; Gene delivery; Nanomedicine; Pharmacokinetics; RNA interference