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.
The dissolution of highly aggregated polyelectrolyte complex particles formed in water after addition
of salt was studied. The dissolution of aggregates proceeded to soluble complexes on the molecular level
of the long-chain component. The driving force of the process is the polyelectrolyte exchange reaction
between the aggregates and the free long chains in excess. The kinetics of the process was studied by
different light scattering techniques. The rate of dissolution showed a strong dependence on the salt
concentration in the solution and on the concentration of the species. The dependence on concentration
of the species in solution weakened with increasing salt concentration. Investigations of the structural
changes during the dissolution process revealed the presence of only two generations of particles in
solution: aggregates and soluble complexes. While the scattering intensity decreased strongly, the
dimensions of the aggregates changed only slightly during dissolution, indicating a spontaneous
disaggregation of the particles. A mechanism of the dissolution process was proposed, which is in agreement
with the experimental findings and previous results in the literature. The process represents a two-step
reaction: The first step consists of the release of the short-chain component from the aggregates by an
exchange reaction via the free long-chain component in solution (second-order reaction). The second step
is the destruction of the aggregates by increasing osmotic pressure in the particle (first-order reaction).
The dissolution process may be understood as a model process for the release of DNA from polyelectrolyte
complexes in gene therapy.
Modification of the polycationic carrier PLL with DMMAn-masked melittin not only enhances gene transfer efficiency, but also strongly reduces the acute toxicity of melittin and PLL. Hence this modification might be useful for optimizing polycationic gene carriers.
RNA interference is a promising therapeutic strategy for treatment of diseases, in particular, cancer. Despite a huge number of targets identified for different cancer types, there are no effective delivery strategies available so far. Polymeric delivery vehicles are often based on large macromolecules. Such approaches often lead to accumulation of toxicity and narrow therapeutic windows. In the current paper, an alternative approach is presented. Low molecular weight oligoethylenimine (OEI) 800 Da was hydrophobically modified through the Michael addition of different alkyl acrylates. An optimal structure containing ten hexyl acrylate residues per one OEI chain (OEI-HA-10) was found to be a promising candidate for siRNA delivery. Hydrophobic modification stabilized the siRNA polyplex structure, increased the colloidal stability of the nanoparticles, and provided lytic properties to OEI required for crossing cellular membranes in the delivery process. In addition, the acrylate ester bond enables fast degradation of OEI-HA-10 into far less toxic components. Further improvement of biological properties of the OEI-HA-10 polyplexes by different formulation strategies was demonstrated. In particular, a remarkable increase of biocompatibility without loss of efficiency could be achieved by coformulation of OEI-HA-10 with lauryl acrylate modified OEI-LA-5.
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