This study presents an efficient method to remove free PEI from PEI polyplexes by SEC. Our results from transfection experiments demonstrate that free PEI substantially contributes to efficient gene expression but also mediates toxic effects in a dose-dependent manner. Purified polyplexes without free PEI have to be applied at increased concentrations to achieve high transfection levels, but exhibit a greatly improved toxicity profile.

Understanding cellular uptake and intracellular processing of nonviral gene delivery systems is a key aspect in developing more efficient vectors. In this study, the impact of clathrin- and caveolae/lipid-raft-dependent endocytosis on cell entry and overall transfection efficiency of polyethylenimine (PEI) polyplexes was evaluated. Most remarkably, the internalization pathway mediating successful transfection depended on both cell type and polyplex type applied. Colocalization studies with transferrin and cholera toxin B revealed that at least two specific endocytosis pathways--the clathrin-dependent and the lipid-raft-dependent--mediated cellular uptake of PEI polyplexes. With the help of specific uptake inhibitors (chlorpromazine and filipin III), cell-line-dependent variations regarding the route of successful transfection were observed (HUH-7, COS-7, HeLa). In COS-7 cells, the clathrin-dependent pathway was the main contributor to the transfection process. In HUH-7 cells, gene transfer by linear PEI polyplexes succeeded mainly via the clathrin-dependent route, whereas transfection by branched PEI polyplexes was mediated by both pathways. In HeLa cells, both pathways were able to mediate successful gene delivery. However, the lipid-raft-dependent pathway was more relevant. The study also revealed that the concentration window between specific inhibitory function and nonspecific toxicity of the uptake inhibitors was very narrow.

The epidermal growth factor receptor (EGFR) is overexpressed on a high percentage of human carcinomas. EGFR is an attractive therapeutic target for tissue-specific targeting by non-viral vectors in cancer gene therapy. In this study we analyzed and compared the effects of EGFR-targeted and untargeted polyplexes in respect to internalization into EGFR overexpressing HuH7 cells. Uptake kinetics and internalization dynamics were evaluated by flow cytometry and single-particle tracking. Our results clearly show that EGFR targeting leads to faster and more efficient internalization compared with untargeted particles. After 5 minutes 50% of the EGFR-targeted polyplexes were internalized, whereas untargeted polyplexes reached only approximately 20% internalization even after 20 minutes. In addition, single-particle tracking revealed a three-phase dynamics of the internalization process, and this was generally observed for polyplexes independent of targeting. Phase I was characterized by slow, actin cytoskeleton-mediated movement of the particles with drift, and included the internalization process. During phase II particles displayed increased velocities with normal and confined diffusion in the cytoplasm. Phase III was characterized by fast active transport along microtubules. Targeting of polyplexes for receptor-mediated endocytosis by the EGFR resulted in shortening of phase I and strongly accelerated internalization.

Tracking transport: Cationic polyethylenimine nanoparticles serve as nonviral vectors and deliver DNA to the nucleus of cells. Fluorescence live‐cell imaging highlights the interactions between these polyplexes and the cytoskeleton during the subsequent stages of transfection (see picture). The analysis of the underlying transport processes gives new impulse to further chemical‐synthesis approaches.