title: Modified poly(propylene imine) dendrimers as effective transfection agents for catalytic DNA enzymes (DNAzymes).
abstract: The major bottleneck in gene therapy remains the issue of delivery. In this work, various modified poly(propylene imine) (PPI) dendrimers are introduced as gene transfection agents. Commercially available PPI-dendrimers have been modified (i) at the exterior primary amines with acetyl groups or glycol gallate (PEG-like) groups, and (ii) at the interior tertiary amines with methyl iodide (MeI) or MeCl to produce multiple quaternized cationic sites in the core of the dendrimer. The prepared materials have been tested with respect to their binding capabilities to DNA, their toxicity in cell cultures, their in vitro transfection efficiency and their in vivo delivery possibilities. In all cases, a 33-mer oligonucleotide (DNAzyme) was used. Polyacrylamide gel electrophoresis (PAGE) studies have demonstrated strong but reversible binding, where the quarternized and higher generation dendrimer species have shown more potent binding. Typically, for the modified fourth PPI-dendrimers, binding is observed at a concentration of about 4 microM DNA and a dendrimer-DNA charge ratio of around 2:1-1:1. All the tested PPI-dendrimers display a low cellular toxicity, especially when higher serum contents are used in the culture medium. For example, most of the prepared fourth generation PPI-dendrimers are not or hardly toxic up to at least 20 microM in 20% serum. An in vitro characterization has revealed a high dendrimer-mediated intracellular uptake of the DNAzyme: all the tested fourth generation PPI-dendrimers display transfection efficiencies close to or exceeding 80%, even when the concentration of serum in the medium is increased from 10 to 40%. Finally, the potential of using modified PPI-dendrimers for in vivo gene therapy experiments is demonstrated. Injecting a G4-PEG(MeI)-ssDNA complex intravenously into Nude mice has resulted in a high nuclear uptake as confirmed by co-localization studies.
authors.affiliation: Erasmus University, Department of Experimental Surgical Oncology, Rotterdam, The Netherlands.
authors.forename: Frederik
authors.initials: F
authors.lastname: Tack
authors.forename: Annette
authors.initials: A
authors.lastname: Bakker
authors.forename: Sophie
authors.initials: S
authors.lastname: Maes
authors.forename: Nathalie
authors.initials: N
authors.lastname: Dekeyser
authors.forename: Monique
authors.initials: M
authors.lastname: Bruining
authors.forename: Cristina
authors.initials: C
authors.lastname: Elissen-Roman
authors.forename: Michel
authors.initials: M
authors.lastname: Janicot
authors.forename: Marcus
authors.initials: M
authors.lastname: Brewster
authors.forename: Henk M
authors.initials: HM
authors.lastname: Janssen
authors.forename: B F M
authors.initials: BF
authors.lastname: De Waal
authors.forename: P M
authors.initials: PM
authors.lastname: Fransen
authors.forename: X
authors.initials: X
authors.lastname: Lou
authors.forename: E W
authors.initials: EW
authors.lastname: Meijer
mesh_terms: D000818:Animals; D002384:Catalysis; D045744:Cell Line, Tumor; D021881:DNA, Catalytic / Q000008:administration & dosage* / Q000235:genetics*; D004337:Drug Carriers / Q000737:chemistry*; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D051379:Mice; D011126:Polypropylenes / Q000737:chemistry*; D014162:Transfection / Q000379:methods*
chemical_list: D021881:DNA, Catalytic; D004337:Drug Carriers; D011126:Polypropylenes; C443641:poly(propyleneimine)