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.

Anionic and cationic block copolymers containing different fractions and chain lengths of poly(ethylene glycol) (PEG) blocks were synthesized and characterized. Polyelectrolyte complex (PEC) formation between these copolymers and homopolyelectrolytes and the stability of these complexes in salt solutions were studied by static and dynamic light scattering and laser Doppler electrophoresis. Complex formation led to particle systems with a high level of aggregation. For the anionic copolymers, even a low content of the short PEG block (M ) 2 kDa) stabilized the PECs near the 1:1 mixing ratio. Independent of the content of the neutral block, the stability of complexes with short PEG blocks with regard to subsequent addition of salt was very low. When copolymers with long PEG blocks were used in PEC formation, high salt stability was observed.

The objective of this work was to obtain gene delivery vectors with high efficiency induced by application of local hyperthermia. As a building construct for the polyplex particles, block copolymers were used, in which one block represents poly(ethyleneimine) (PEI) and another block a statistical copolymer of poly(N-isopropylacryamide) (PNIPAM) and different hydrophilic monomers (acrylamide or vinylpyrrolidinone). The block copolymers were synthesizized by radical polymerization of the corresponding monomers directly onto PEI. The complexation of DNA with these copolymers led to small, charge neutral particles, which aggregated upon increasing the temperature from 37 degrees C to 42 degrees C. This aggregation was found to be responsible for the enhanced transfection efficiency of these formulations under hyperthermic conditions. Gene expression in cells treated by hyperthermia was found to be nearly 2 orders of magnitude higher in comparison to cells transfected at physiological temperature. The mechanism by which hyperthermia influences the gene transfection efficiency is proposed.

Nanoparticles made of polylactide-poly(ethylene glycol) block-copolymer (PLA-PEG) are promising vehicles for drug delivery due to their biodegradability and controllable payload release. However, published data on the drug delivery properties of PLA-PEG nanoparticles are heterogeneous in terms of nanoparticle characteristics and mostly refer to low injected doses (a few mg nanoparticles per kg body weight). We have performed a comprehensive study of the biodistribution of nanoparticle formulations based on PLA-PEG nanoparticles of ~100nm size at injected doses of 30 to 140mg/kg body weight in healthy rats and nude tumor-bearing mice. Nanoparticle formulations differed by surface PEG coverage and by release kinetics of the encapsulated model active pharmaceutical ingredient (API). Increase in PEG coverage prolonged nanoparticle circulation half-life up to ~20h in rats and ~10h in mice and decreased retention in liver, spleen and lungs. Circulation half-life of the encapsulated API grew monotonously as the release rate slowed down. Plasma and tissue pharmacokinetics was dose-linear for inactive nanoparticles, but markedly dose-dependent for the model therapeutic formulation, presumably because of the toxic effects of released API. A mathematical model of API distribution calibrated on the data for inactive nanoparticles and conventional API form correctly predicted the distribution of the model therapeutic formulation at the lowest investigated dose, but for higher doses the toxic action of the released API had to be explicitly modelled. Our results provide a coherent illustration of the ability of controllable-release PLA-PEG nanoparticles to serve as an effective drug delivery platform to alter API biodistribution. They also underscore the importance of physiological effects of released drug in determining the biodistribution of therapeutic drug formulations at doses approaching tolerability limits.

The knowledge of the biodistribution of macromolecular drug formulations is a key to their successful development for specific tissue- and tumor-targeting after systemic application. Based on the polyplex formulations, we introduce novel drug nanocarriers, which we denote as "quantoplexes" incorporating near-infrared (IR)-emitting cadmium telluride (CdTe) quantum dots (QDs), polyethylenimine (PEI), and a macromolecular model drug [plasmid DNA (pDNA)], and demonstrate the ability of tracking these bioactive compounds in living animals. Intravenous application of bare QD into nude mice leads to rapid accumulation in the liver and peripheral regions resembling lymph nodes, followed by clearance via the liver within hours to days. Quantoplexes rapidly accumulate in the lung, liver, and spleen and the fluorescent signal is detectable for at least a week. Tracking quantoplexes immediately after intravenous injection shows rapid redistribution from the lung to the liver within 5 minutes, depending on the PEI topology and quantoplex formulation used. With polyethyleneglycol (PEG)-modified quantoplexes, blood circulation and passive tumor accumulation was measured in real time. The use of quantoplexes will strongly accelerate the development of tissue and tumor-targeted macromolecular drug carriers.