Polymer carriers like PEI which proved their efficiency in DNA delivery were found to be far less effective for the applications with siRNA. In the current study, we generated a number of nontoxic derivates of branched PEI through modification of amines by ethyl acrylate, acetylation of primary amines, or introduction of negatively charged propionic acid or succinic acid groups to the polymer structure. The resulting products showed high efficiency in siRNA-mediated knockdown of target gene. In particular, succinylation of branched PEI resulted in up to 10-fold lower polymer toxicity in comparison to unmodified PEI. Formulations of siRNA with succinylated PEI were able to induce remarkable knockdown (80% relative to untreated cells) of target luciferase gene at the lowest tested siRNA concentration of 50 nM in Neuro2ALuc cells. The polyplex stability assay revealed that the efficiency of formulations which are stable in physiological saline is independent of the affinity of siRNA to the polymer chain. The improved properties of modified PEI as siRNA carrier are largely a consequence of the lower polymer toxicity. In order to achieve significant knockdown of target gene, the PEI-based polymer has to be applied at higher concentrations, required most probably for sufficient accumulation and proton sponge effects in endosomes. Unmodified PEI is highly toxic at such polymer concentrations. In contrast, the far less toxic modified analogues can be applied in concentrations required for the knockdown of target genes without side effects.

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Extracellular stability of electrostatically formed siRNA polyplexes is a significant concern in the delivery process. To overcome the risk of polyplex dissociation in the extracellular environment, siRNA was covalently incorporated into a pH- and redox-responsive polymer conjugate. The novel siRNA conjugate consists of polylysine (PLL) as RNA binding and protecting polycation, polyethylene glycol (PEG) as solubilizing and shielding polymer, the lytic peptide melittin masked by dimethylmaleic anhydride (DMMAn) removable at endosomal pH, and the siRNA attached at the 5'-end of the sense strand via a bioreducible disulfide bond. The purified siRNA conjugate was stable in the presence of the polyanion heparin at conditions where the analogous electrostatic siRNA polyplexes disassemble. Only the combination of heparin plus a reducing agent such as glutathione triggered the release of siRNA from the conjugate. High in vitro biocompatibility (absence of cytotoxicity or hemolytic activity at neutral pH) and efficient and sequence-specific gene silencing was found at > or =25 nM siRNA, comparable to the corresponding electrostatic polyplexes. In vivo toxicity studies of this formulation demonstrated that conjugates remain to be optimized for therapeutic application.