[Show abstract][Hide abstract] ABSTRACT: The pseudodendritic, biodegradable polymer HD-O, consisting of an OEI800 core with several OEI800 molecules attached to it via 1.6-hexanediol diacrylate linkers, has potent pDNA but poor siRNA delivery ability, due to instability of the resulting siRNA polyplexes. Stabilization of such nanoparticles by crosslinking surface amines of HD-O in the polyplexes with dithiobis-(succinimidylpropionate) (DSP) greatly enhanced gene silencing efficiency. Successful crosslinking on the polyplex surface was indicated by a decrease of the positive Zeta potential of the polyplexes. Tuning the polymer/siRNA ratio in combination with adjustment of the linker to a molar ratio of 0.05/1 between linker and polymer amines proved essential for transfection efficiency and prevention of particle aggregation. Gene silencing ability of the crosslinked particles was demonstrated in murine neuroblastoma N2A and human hepatoma HUH-7 cells. Flow cytometry showed efficient cellular uptake already after 1 h incubation with the crosslinked but not with unstabilized particles. Downregulation of endogenous AHA1 mRNA (85% knockdown compared to control) by crosslinked HD-O/AHA1-siRNA particles was detected by quantitative real-time PCR.
European journal of pharmaceutical sciences: official journal of the European Federation for Pharmaceutical Sciences 12/2012; 47(5):914–920. DOI:10.1016/j.ejps.2012.09.006 · 3.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cationic oligomers were assembled by solid-phase supported synthesis in few coupling steps based on C-terminal alanine and two lysine branchings, followed by elongation of the four arms with two to five repeats of artificial oligoamino acids containing the 1,2-diaminoethane motif, and ended by N-terminal cysteines or alanines. These sequence-defined oligomers, containing between 28 and 68 protonatable nitrogens, were evaluated for complex formation with plasmid DNA (pDNA) and short interfering RNA (siRNA), followed by reporter gene transfer and gene silencing experiments in Neuro2A cells. By two simple variations, the pDNA gene transfer activity could be thousand-fold improved, exceeding the gold standard linear PEI up to >50-fold. Firstly, the N-terminal cysteines introduced for bioreversible stabilization of polyplexes by internal disulfide links after complex formation greatly enhanced gene transfer. Secondly, variation of the artificial oligoamino acid building blocks containing either triethylene tetramine (Gtt), tetraethylene pentamine (Stp), or pentaethylene hexamine (Sph) disclosed a clear ranking in the order of Sph>Stp>Gtt for both pDNA compaction and transfection activity. Extending the chain lengths of the arms beyond three building blocks had marginal impact on the performance. For the much smaller siRNA cargo, polyplex stabilization by cysteine disulfides presents a strict requirement. Sph and Stp based cysteine-ended four-arms displayed similar binding activity, with Stp providing best gene silencing efficiency.
[Show abstract][Hide abstract] ABSTRACT: Although our understanding of RNAi and our knowledge on designing and synthesizing active and safe siRNAs significantly increased during the past decade, targeted delivery remains the major limitation in the development of siRNA therapeutics. On one hand, practical considerations dictate robust chemistry reproducibly providing precise carrier molecules. On the other hand, the multistep delivery process requires dynamic multifunctional carriers of substantial complexity. We present a monodisperse and multifunctional carrier system, synthesized by solid phase supported chemistry, for siRNA delivery in vitro and in vivo. The sequence-defined assembly includes a precise cationic (oligoethanamino)amide core, terminated at the ends by two cysteines for bioreversible polyplex stabilization, at a defined central position attached to a monodisperse polyethylene glycol chain coupled to a terminal folic acid as cell targeting ligand. Complexation with an endosomolytic influenza peptide-siRNA conjugate results in nanosized functional polyplexes of 6 nm hydrodynamic diameter. The necessity of each functional substructure of the carrier system for a specific and efficient gene silencing was confirmed. The nanosized polyplexes showed stability in vivo, receptor-specific cell targeting, and silencing of the EG5 gene in receptor-positive tumors. The nanosized appearance of these particles can be precisely controlled by the oligomer design (from 5.8 to 8.8 nm diameter). A complete surface charge shielding together with the high stability result in good tolerability in vivo and the absence of accumulation in nontargeted tissues such as liver, lung, or spleen. Due to their small size, siRNA polyplexes are efficiently cleared by the kidney.
[Show abstract][Hide abstract] ABSTRACT: Sequence defined oligo (ethane amino) amides produced by solid-phase supported synthesis using different building blocks and molecular shapes were tested for structure-activity relationships in siRNA delivery. Efficient reporter gene knockdown was obtained in a variety of cell lines using either branched three-armed structures, or lipid-modified structures with i-shape, T-shape, U-shape configuration. For the majority of structures (apart from U-shapes), the presence of 2 or 3 cysteines was strictly required for polyplex stabilization and silencing activity. Although all four building blocks contain the ethylenediamine proton sponge motif, only oligomers assembled with the tetraethylenepentamine based amino acids (Stp, Gtp, Ptp) but not with the triethylenetetramine based amino acid (Gtt) were able to mediate efficient gene silencing. For the lipopolymeric structures, out of the tested saturated (from C4 to C18) and unsaturated (C18) fatty acid moieties, two proximate oleic acids or linolic acids provided the oligomers with the best gene silencing activity and also pH specific lytic activity at pH 5.5, presumably facilitating endosomal escape of the polyplexes. Evaluation of oligomer chain length revealed a minimal number of at least two oligo (ethane amino) building blocks per oligomer arm as necessary for the vast majority of structures, but only marginal changes were found with higher numbers (structures with up to 60 ethane amino nitrogens were evaluated). Two promising carriers (T-shape 49, i-shape 229) were also evaluated for EG5 siRNA delivery. This resulted in tumor cell cycle arrest, and appearance of mitotic monoastral spindles both in vitro and in vivo upon systemic delivery. Repeated intratumoral treatment with EG5 siRNA polyplexes significantly reduced Neuro2A-eGFPLuc tumor growth in a siRNA-specific manner.
[Show abstract][Hide abstract] ABSTRACT: Künstliche Oligoethylenaminosäuren wurden zusammen mit natürlichen Aminosäuren und optional Fettsäuren zur Festphasen-basierten Synthese von Polymeren mit präziser Sequenz, Topologie und Modifikation eingesetzt. Erste Konzeptstudien demonstrieren das große Potenzial solcher Polymere in der Transfektion von pDNA und siRNA.
[Show abstract][Hide abstract] ABSTRACT: Artificial oligo(ethylene amino) acids, together with selected natural amino acids and fatty acid modifications, have been used for the solid-phase-assisted synthesis of polymers with precise sequence, topology, and modifications (see picture). First proof-of-concept studies demonstrate the high potential of such polymers in pDNA and siRNA delivery. K=lysine, A=alanine.
[Show abstract][Hide abstract] ABSTRACT: HD O is a low molecular weight pseudodendrimer containing oligoethylenimine and degradable hexanediol diacrylate diesters. DNA polyplexes display encouraging gene transfer efficiency in vitro and in vivo but also a limited stability under physiological conditions. This limitation must be overcome for further development into more sophisticated formulations.
HD O polyplexes were laterally stabilized by crosslinking surface amines via bifunctional crosslinkers, bioreducible dithiobis(succimidyl propionate) (DSP) or the nonreducible analog disuccinimidyl suberate (DSS). Optionally, in a subsequent step, the targeting ligand transferrin (Tf) was attached to DSP-linked HD O polyplexes via Schiff base formation between HD O amino groups and Tf aldehyde groups, which were introduced into Tf by periodate oxidation of the glycosylation sites.
Crosslinked DNA polyplexes showed an increased stability against exchange reaction by salt or heparin. Disulfide bond containing DSP-linked polyplexes were susceptible to reducing conditions. These polyplexes displayed the highest gene expression levels in vitro and in vivo (upon intratumoral application in mice), and these were significantly elevated and prolonged over standard or DSS-stabilized HD O formulations. DSP-stabilized HD O polyplexes with or without Tf coating were well-tolerated after intravenous application. High gene expression levels were found in tumor tissue, with negligible gene expression in any other organ.
Lateral stabilization of HD O polyplexes with DSP crosslinker enhanced gene transfer efficacy and was essential for the incorporation of a ligand (Tf) into a stable particle formulation.
The Journal of Gene Medicine 02/2010; 12(2):180-93. DOI:10.1002/jgm.1430 · 2.47 Impact Factor