title: Removable nanocoatings for siRNA polyplexes.
abstract: To assist in overcoming the inherent instability of nucleic acid-containing polyplexes in physiological solutions, we have here set out to develop removable nanocoatings for modifying the surface of siRNA-based nanoparticles. Here, N-(2-hydroxypropyl)methacrylamide (HPMA) based copolymers containing carbonylthiazolidine-2-thione (TT) reactive groups in their side chains bound via disulfide spacers to the polymeric backbone were synthesized, and these copolymers were used to coat the surface of polyplexes formed by the self-assembly of anti-Luciferase siRNA with the polycations polyethylene imine (PEI) and poly(HPMA)-grafted poly(l-lysine) (GPL). The coating process was monitored by analyzing changes in the weight-average molecular weight (M(w)), the hydrodynamic radius (R(h)), and the zeta-potential (ζ) of the polyplexes, using both static (SLS) and dynamic (DLS) light scattering methods. The outlined methods resulted in the attachment of, on average, 28 polymer molecules to the surface of the polyplexes, forming a ∼5-nm-thick hydrophilic stealth coating. Initial efforts to develop RGD-targeted coated polyplexes are also described. Atomic force microscopy (AFM) showed an angular polyplex structure and confirmed the narrow size distribution of the coated nanoparticles. The stability of the polymer-coated and uncoated polyplexes was evaluated by gel electrophoresis and by turbidity measurements, and it was found that modifying the surface of the siRNA-containing polyplexes substantially improved their stability in physiological solutions. Using polymer-coated GPL-based polyplexes containing anti-Luciferase siRNA, we finally also obtained some initial proof-of-principle for time- and concentration-dependent target-specific gene silencing, suggesting that these systems hold significant potential for further in vitro and in vivo evaluation.
authors.affiliation: Institute of Macromolecular Chemistry, v. v. i., Academy of Sciences of the Czech Republic, Heyrovský sq. 2, 162 06, Prague 6, Czech Republic. kostka@imc.cas.cz
authors.forename: Libor
authors.initials: L
authors.lastname: Kostka
authors.forename: Cestmír
authors.initials: C
authors.lastname: Konák
authors.forename: Vladimír
authors.initials: V
authors.lastname: Subr
authors.forename: Milena
authors.initials: M
authors.lastname: Spírková
authors.forename: Yoseph
authors.initials: Y
authors.lastname: Addadi
authors.forename: Michal
authors.initials: M
authors.lastname: Neeman
authors.forename: Twan
authors.initials: T
authors.lastname: Lammers
authors.forename: Karel
authors.initials: K
authors.lastname: Ulbrich
mesh_terms: D004220:Disulfides / Q000737:chemistry; D015394:Molecular Structure; D053758:Nanoparticles / Q000737:chemistry*; D011108:Polymers / Q000138:chemical synthesis / Q000737:chemistry*; D034741:RNA, Small Interfering / Q000737:chemistry*; D013237:Stereoisomerism; D013499:Surface Properties; D013871:Thiones / Q000737:chemistry
chemical_list: D004220:Disulfides; D011108:Polymers; D034741:RNA, Small Interfering; D013871:Thiones