Polyethylenimine-based nucleic acid delivery

With recent advances in molecular biology and the sequencing of the human genome, nucleic acids are expected to engage a pivotal position in the development of novel therapeutic concepts. Although viral vectors are very effective and are capable of many of the required tasks for efficient gene transfer, their broad use is limited by severe safety risks [1,2]. Therefore, this work focused on the use of polymers as non-viral vehicles for nucleic acid delivery. Among various polymers, polyethylenimine (PEI) has become a gold standard for nucleic acid delivery [3]. The relatively high transfection efficiency of branched PEI (BPEI) -based delivery systems is seriously afflicted by the cytotoxicity of the polymer. In contrast, the linear form (LPEI) has a particularly high gene expression accompanied by a lower cytotoxicity, but nevertheless the transfection efficiency of LPEI 22 kDa (ExGen®) cannot be enhanced beyond a certain limit due to cytotoxicity. Therefore, the ultimate goal of this thesis was to identify strategies towards effective PEI-based delivery systems with low cytotoxicity for in vitro use. The first objective was to overcome the frequent restriction that high transfection efficiency is limited by the cytotoxicity of the non-viral carrier. We explored the potential of utilizing LPEIs with a molecular weight (MW) ranging from 1.0 to 9.5 kDa to overcome this limitation (Chapter 2). This study envisioned the decoupling of transfection efficiency from cytotoxicity and demonstrated that LPEIs with low MW are more efficient and significantly less toxic than their high MW counterparts in CHO-K1 cells in vitro. A follow-up study focused on the uptake and intracellular stability of various LPEI - polyplexes and plasmid DNA in order to investigate differences relevant for their efficacy. Furthermore, the influence of the ionic strength of the polyplex formation medium and presence of serum during the transfection process on the transfection efficiency was evaluated (Chapter 3). The results showed that we have a very robust system for LPEI-based nucleic acid delivery that is widely independent of external influences and suitable for routine transfection experiments. The interaction of polymer and plasmid DNA in living cells was additionally evaluated by fluorescence resonance energy transfer (FRET) -based techniques (Chapter 4). Furthermore, we hypothesized that a bioreversible crosslinking of low MW LPEIs would raise the polymer�s efficacy due to the higher MW, while the biodegradable linkages would undergo intracellular breakdown and hence minimize toxicity (Chapter 5). Our results proved the hypothesis: the most effective biodegradable PEI (LR-PEI) was capable of gene delivery in 27-fold more cells than the non-degradable starting material and moreover, the maximum transfection efficiency achieved in CHO-K1 cells had a value of about 73%, which is much higher than that of the LPEI derivative of similar MW. These biodegradable PEIs can be counted among the most effective polymer-based gene delivery vehicles, because the efficacy was nearly 3-fold higher than with BPEI 25 kDa and LPEI 22 kDa, to which newly synthesized polymers are usually compared. LR-PEI-mediated gene delivery was dependent on intracellular reduction and could be modulated by manipulation of the number of stabilizing bonds. After biodegradable PEI-based polyplexes had successfully been applied for the transfection of various cell lines in vitro, the next step was to evaluate their gene transfer ability in human primary cells and in a �hard-to-transfect� cell line (Chapter 6). In dendritic cells the transfection efficiency was nearly negligible and also accompanied by a remarkable toxicity. Human chondrocytes were more susceptible to PEI-based transfection, but the efficacy was much lower compared to the cell lines tested in Chapter 5. Last mentioned, we showed that the PEI backbone is a necessary prerequisite for efficient polymer-based gene delivery by the investigation of per-N-methylated PEI (Chapter 7). References: 1. Marshall E. Science 2002; 298: 510-511. 2. Sadelain M. Gene Ther 2004; 11: 569-573. 3. Boussif O, Lezoualc'h F, Zanta MA, Mergny MD, Scherman D, Demeneix B, Behr J. Proc Natl Acad Sci U S A 1995; 92: 7297-7301