Oligonucleotides such as short, double-stranded RNA (siRNA) or plasmid DNA (pDNA) promise high potential in gene therapy. For pharmaceutical application, however, adequate drug carriers are required. Among various concepts progressing in the market or final development, nanosized hydrogel particles may serve as novel transport media especially for siRNA. In this work, a new concept of synthesizing polymeric cationic nanohydrogels was developed, which offers a promising strategy to complex and transport siRNA into cells. For this purpose, amphiphilic reactive ester block copolymers were synthesized by RAFT polymerization of pentafluorophenyl methacrylate as reactive ester monomer together with tri(ethylene glycol)methyl ether methacrylate. In polar aprotic solvents, a self-assembly of these polymers could be observed leading to the formation of nanometer-sized polymer aggregates. The resulting superstructures were used to convert the reactive precursor block copolymers with amine-containing cross-linker molecules into covalently stabilized hydrogel particles. Detailed dynamic light scattering studies showed that the structure of the self-assembled aggregates can permanently be locked-in by this process. This method offers a new possibility to synthesize precise nanohydrogels of different size starting from various block copolymers. Moreover, via reactive ester approach, further functionalities could be attached to the nanoparticle, such as fluorescent dyes, which allowed distinct tracing of the hydrogels during complexation with siRNA or cell uptake experiments. In this respect, cellular uptake of the particles themselves as well as with its payload could be detected successfully. Looking ahead, these novel cationic nanohydrogel particles may serve as a new platform for proper siRNA delivery systems.

Here, the preparation of a novel block copolymer consisting of a statistical copolymer N-(2-hydroxypropyl) methacrylamide-s-N-(3-aminopropyl) methacrylamide and a short terminal 3-guanidinopropyl methacrylamide block is reported. This polymer structure forms neutral but water-soluble nanosized complexes with siRNA. The siRNA block copolymer complexes are first analyzed using agarose gel electrophoresis and their size is determined with fluorescence correlation spectroscopy. The protective properties of the polymer against RNA degradation are investigated by treating the siRNA block copolymer complexes with RNase V1. Heparin competition assays confirm the efficient release of the cargo in vitro. In addition, the utilization of microscale thermophoresis is demonstrated for the determination of the binding strength between a fluorescently labeled polyanion and a polymer molecule.

A propargyl containing guanosine phosphoramidite was synthesized and incorporated into siRNA, enabling click-ligation with an azido fluorophore onto the nucleobase sugar edge. Duplex stability was not affected by labeling at this new site, which allowed deconvolution of the effects of label, structure and attachment site on RNAi activity.

We introduce new methods for the efficient and informative characterization of siRNA polyplexes with special attention to stability. Comparing FRET-labeled siRNA in different polyplexes associates successful knockdown with intermediate siRNA stability in various steps from formulation to intracellular persistence.