Imidazole and dimethyl aminopropyl-functionalized hyperbranched polymers for nucleic acid transfection.

In order to mimic the histidine binding motives of naturally occurring histones as DNA complexing proteins, hyperbranched poly(ethylene imine) and polyglycerol were functionalized with imidazole or 3-dimethylamino propyl groups. These new polycationic polymers were tested for interaction with dye-labelled oligonucleotide and DNA using UV and fluorescence spectroscopy and gel electrophoresis. Formation of stable complexes was observed above N/P ratios of 4 for unfunctionalized and 8 for functionalized PEIs. No stable complexes were formed with polyglycerol-based polyamines up to N/P 16. Cytotoxicity determined by MTT assay of all functionalized PEI polymers was found to be significantly lower than for unfunctionalized PEI. PG-based polymers showed no toxicity in the tested concentration range. Dynamic light scattering showed that only for PEI(21)-Imidaz polyplexes hydrodynamic diameters below 250 nm could be reached.The influence of functionalization and polymer type on transfection efficiency was evaluated in L929, NIH/3T3 and HeLa cells. Only imidazole-functionalized PEIs reached similar transfection efficiencies as unfunctionalized PEIs, while 3-dimethylamino propyl modification resulted in lower transfection efficiencies. We also demonstrated that the polymer plays an important role for transfection properties since, regardless of the modifications of polyglycerol, only low transfection efficiencies were observed at functionalization levels below 50%.

Insights into effective RNAi gained from large-scale siRNA validation screening.

Transfection of chemically synthesized short interfering RNAs (siRNAs) enables a high level of sequence-specific gene silencing. Although siRNA design algorithms have been improved in recent years, it is still necessary to prove the functionality of a given siRNA experimentally. We have functionally tested several thousand siRNAs for target genes from various gene families including kinases, phosphatases, and cancer-related genes (e.g., genes involved in apoptosis and the cell cycle). Some targets were difficult to silence above a threshold of 70% knockdown. By working with one design algorithm and a standardized validation procedure, we discovered that the level of silencing achieved was not exclusively dependent on the siRNA sequences. Here we present data showing that neither the gene expression level nor the cellular environment has a direct impact on the knockdown which can be achieved for a given target. Modifications of the experimental setting have been investigated with the aim of improving knockdown efficiencies for siRNA-target combinations that show only moderate knockdown. Use of higher siRNA concentrations did not change the overall performance of the siRNA-target combinations analyzed. Optimal knockdown at the mRNA level was usually reached 48-72 hours after transfection. Target gene-specific characteristics such as the accessibility of the corresponding target sequences to the RNAi machinery appear to have a significant influence on the knockdown observed, making certain targets easy or difficult to knock down using siRNA.