Mechanistic insights into linear polyethylenimine-mediated gene transfer.

We recently debuted a variety of linear polyethylenimines (LPEIs) with low molecular weight as carriers for gene delivery. The highest transfection efficiency (approximately 44%) was obtained with LPEI 6.6 kDa, while the cytotoxicity remained low (approximately 90% of CHO-K1 cells survived the transfection procedure). Here, we investigated various steps during the transfection process using LPEI 8.1, 5.0 and 1.8 kDa, in order to gain a more complete insight into LPEI-mediated gene transfer and to explore conceptual aspects for further optimization. The cellular uptake characterized by flow cytometry was similar for LPEI 8.1 and 5.0 kDa, while it was significantly lower for LPEI 1.8 kDa. The transfection efficacy in contrast was at NP 24 20.07% for LPEI 8.1 kDa and 39.71% for LPEI 5.0 kDa. This suggests that the endocytosis seems not to be a decisive parameter that determines the efficacy of a polymer in the transfection process. Real-time PCR investigations revealed that LPEI 1.8 kDa likewise or even better protected plasmid from degradation compared to LPEI 5.0 or 8.1 kDa. Furthermore, we found that 1/6 to 1/3 intact plasmid DNA reached the intracellular compartments after complexation with LPEI 1.8 kDa. Therefore, the amount of plasmid DNA available in the cytoplasm seems not to be a limiting factor in the transfection process. That LPEI 8.1-polyplexes built at NP 12 in glucose and transfected in serum-free culture conditions were superior to those built in sodium chloride or transfected in serum-containing conditions points at the structure as a decisive parameter deserving more attention in future studies.

Fluorescence resonance energy transfer: evaluation of the intracellular stability of polyplexes.

The investigation of intracellular mechanisms of non-viral nucleic acid delivery systems has provided great impetus for the improvement of their efficacy. Especially the intracellular release of the nucleic acid from the non-viral carrier system may be a relevant criterion for the high transfection efficiency of certain polymers. Therefore, we evaluated fluorescence resonance energy transfer (FRET) in combination with confocal laser scanning microscopy or flow cytometry as tool to determine the intracellular disintegration of polyplexes built with plasmid DNA and linear polyethylenimine. In microscopy, which allowed for an observation of polyplexes within single cells, sensitized emission measurement and acceptor photobleaching have been tested towards quantitative FRET analysis. In contrast, the whole cell population was analyzed by the flow cytometry-based method. We suggest that FRET is a useful tool to evaluate the intracellular disintegration of polyplexes built with various polymers.

Polyplexes of polyethylenimine and per-N-methylated polyethylenimine-cytotoxicity and transfection efficiency.

For non-viral gene delivery, the carriers for DNA transfer into cells must be vastly improved. The branched cationic polymer polyethylenimine has been described as an efficient gene carrier. However, polyethylenimine was demonstrated to mediate substantial cytotoxicity. Therefore, this study is aimed at investigating per-N-methylated polyethylenimine, which is thought to have a much lower cytotoxicity due to its lower charge density. Results from a gel retardation assay and laser light scattering indicated that per-N-methylated polyethylenimine condenses DNA into small and compact nanoparticles with a mean diameter <150 nm. Furthermore, polyplexes of polyethylenimine and per-N-methylated polyethylenimine with DNA had a positive zeta potential and the polymers protected DNA from nuclease-mediated digestion. The transfection efficiency of polyethylenimine and per-N-methylated polyethylenimine was tested in CHO-K1 cells. Using green fluorescent protein as reporter gene and flow cytometry analysis, we demonstrated that per-N-methylated polyethylenimine has a lower cytotoxicity, but also a significantly lower transfection efficiency. Using propidium iodide staining, we could additionally distinguish between viable and dead cells. At NP > or = 12, per-N-methylated polyethylenimine showed a much higher cell viability and the ratio of viable and transfected cells to dead and transfected cells was about 1.5 to 1.7 fold higher than for polyethylenimine. The results of cell viability from flow cytometry analysis were confirmed by the MTS assay. Using luciferase reporter gene for transfection experiments, the gene expression of per-N-methylated polyethylenimine was lower at NP 6, 12 and 18 as compared to polyethylenimine, but at NP 24 it yielded similar levels.

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.

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.

Gene delivery with low molecular weight linear polyethylenimines.

BACKGROUND: 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. METHODS: Polyplexes of plasmid DNA encoding for the enhanced green fluorescent protein (EGFP) and various LPEIs were compared concerning their transfection efficiency and cytotoxicity in CHO-K1 and HeLa cells by flow cytometry. The involvement of endolysosomes in LPEI-mediated gene transfer was investigated by applying the proton pump inhibitor bafilomycin A1 and the lysosomotropic agent sucrose. Confocal laser scanning microscopy was applied to assess the size and shape of polyplexes under cell culture conditions, to detect their endolysosomal localization and to observe their translocation to the nucleus. RESULTS: The transfection efficiency could be altered by varying the MW and the amount of the polymer available for polyplex formation. The highest transfection efficiency (about 44%), i.e. the fraction of EGFP-positive cells, was obtained with LPEI 5.6 kDa, while the cytotoxicity remained low. The colocalization of polyplexes and endolysosomes was observed, and it appeared that the larger polyplexes escaped from the acidic organelles particularly quickly. For LPEI 5.0 and 9.0 kDa, the number of cells and nuclei that had taken up DNA after 6 hours was similar, as determined by flow cytometry. CONCLUSIONS: Our study suggests that LPEIs with low MWs are promising candidates for non-viral gene delivery, because they are more efficient and substantially less toxic than their higher MW counterparts.