Uta Lungwitz

Breaking up the correlation between efficacy and toxicity for nonviral gene delivery

Nonviral nucleic acid delivery to cells and tissues is considered a standard tool in life science research. However, although an ideal delivery system should have high efficacy and minimal toxicity, existing materials fall short, most of them being either too toxic or little effective. We hypothesized that disulfide cross-linked low-molecular-weight (MW) linear poly(ethylene imine) (MW <4.6 kDa) would overcome this limitation. Investigations with these materials revealed that the extracellular high MW provided outstandingly high transfection efficacies (up to 69.62 ± 4.18% in HEK cells). We confirmed that the intracellular reductive degradation produced mainly nontoxic fragments (cell survival 98.69 ± 4.79%). When we compared the polymers in >1,400 individual experiments to seven commercial transfection reagents in seven different cell lines, we found highly superior transfection efficacies and substantially lower toxicities. This renders reductive degradation a highly promising tool for the design of new transfection materials.

Mechanistic investigation of poly(ethylene imine)-based siRNA delivery: Disulfide bonds boost intracellular release of the cargo

Poly(ethylene imine) (PEI) has gained increasing attention in the delivery of small interfering RNAs (siRNAs) into cells. In order to further optimize PEI for this application, the first goal of this study was to examine particular steps of siRNA delivery with various PEI derivatives as carriers. Furthermore, the hypothesis that disulfide cleavable carrier systems are favorable for the release of siRNA into the cell cytoplasm was investigated. Flow cytometry and confocal microscopy were used to assess the cellular uptake and intracellular distribution of siRNA, which were then related to gene silencing efficacy. We observed a strong correlation between cellular uptake and RNAi activity. The cellular uptake of siRNA was more efficient with increasing branching of the polymer, i.e. linear PEI (lPEI) 5 kDa < lPEI cross-linked via disulfide bonds (ssPEI) < branched PEI (bPEI) 25 kDa. However, it was also evident that the siRNA release from the carrier, which was promoted by ssPEI, played an important role in the accessibility of siRNA for the gene silencing complex. Therefore, we suggest that a combination of a high branching density and reductively cleavable bonds within the PEI-based carrier system could be one possible step towards improving siRNA delivery.

Gene delivery with low molecular weight linear polyethylenimines.

Linear polyethylenimine (LPEI) with a molecular weight (MW) of 22 kDa has been described as having a superior ability to induce gene transfer compared to its branched form. However, the transfection efficiency of the polymer cannot be enhanced beyond a certain limit due to cytotoxicity. We explored the potential of utilizing LPEIs with MWs ranging from 1.0 to 9.5 kDa to overcome this limitation.