Thorsten Bieber

A novel non-viral vector for DNA delivery based on low molecular weight, branched polyethylenimine: effect of molecular weight on transfection efficiency and cytotoxicity.

AbstractPurpose. Low molecular weight branched polyethylenimine (LMW-PEI) was synthesized and studied as a DNA carrier for gene delivery with regard to physico-chemical properties, cytotoxicity, and transfection efficiency. Methods. The architecture of LMW-PEI, synthesized by acid catalyzed ring-opening polymerization of aziridine was characterized by size exclusion chromatography in combination with laser light scattering and 13C-NMR-spectroscopy. In vitro cytotoxic effects were quantified by LDH and MTT assay and visualized by transmission electron microscopy. The potential for transgene expression was monitored in ECV304 cells using luciferase driven by a SV40 promoter as reporter gene system. Results. LMW-PEI (Mw 11′900 D) with a low degree of branching was synthesized as a DNA carrier for gene delivery. In contrast to high molecular weight polyethylenimines (HMW-PEI; Mw l′616′OOO D), the polymer described here showed a different degree of branching and was less cytotoxic in a broad range of concentrations. As demonstrated by transmission electron microscopy the LMW-PEI formed only small aggregates which were efficiently taken up by different cells in the presence of serum, most likely by an endocytic pathway. LMW-PEI yielded transfection efficiencies measured via expression of the reporter gene luciferase which were up to two orders of magnitude higher than those obtained with HMW-PEI. The reporter gene expression was concentration dependent, but in contrast to lipofection independent of serum addition. Conclusions. The LMW-PEI described here is a new, highly efficient, and non-cytotoxic vector with a favorable efficiency/toxicity profile for gene therapeutic applications.

Intracellular route and transcriptional competence of polyethylenimine-DNA complexes

Polyethylenimine (PEI) is a cationic polymer which can be complexed with DNA. PEI-DNA complexes can be used for in vitro and in vivo gene delivery approaches. The excess of positive surface charges enhances the association of the complex with the plasmamembrane of cells and facilitates their uptake by endocytosis. The intracellular transport pathway from the endosome to the nucleus is not understood. Here we show that PEI-DNA complexes are taken up by all cells which are treated with these complexes, indicating, that the uptake is not the rate limiting step in the final transfection efficiency. We reveal by fluorescent microscopy, cell fractionation studies and electron microscopy, that PEI-DNA complexes accumulate in the lysosomal compartment, from where they are released through small local membrane damages. However, the cytoplasmic pool of PEI-DNA complexes is small and with the applied morphological approaches PEI aggregates could not be detected in the nucleus. This indicates, that only a small fraction of the complexes reach their final destiny. To test whether the association of DNA with PEI might be the critical step for transfection, we performed in vitro transcription assays with PEI-DNA complexes. These experiments revealed, that the transcription is not impaired when PEI is closely attached to the template DNA. Our results thus point to the transfer of PEI-DNA complexes from the lysosomal compartment to the nucleus as the rate limiting step in cell transfection.

Cationized human serum albumin as a non-viral vector system for gene delivery? Characterization of complex formation with plasmid DNA and transfection efficiency.

Cationized human serum albumin (cHSA) could serve as a potential non-viral vector system for gene delivery. Native human serum albumin was cationized by covalent coupling of hexamethylenediamine to the carboxyl groups resulting in a shift of the isoelectric point from pH 4-5 to 7-9. The cationized albumin underwent spontaneous self-assembly with DNA as demonstrated by retardation of CMV-nlacZ plasmid in agarose gel electrophoresis. Photon correlation spectroscopy showed a decrease of complex size with increasing cHSA/plasmid ratios. Under optimized conditions complexes were formed with 230-260 nm mean diameter and a homogenous, narrow size distribution. At room temperature complexes were stable in 0.9% sodium chloride solution pH 7.4 for 1 h without aggregation. Process parameters such as albumin concentration, incubation time, temperature, pH, order of reagent addition, the presence of bivalent ions and the ionic strength of the complexation medium all influenced the complex size. Confocal laser scanning microscopy showed interactions of a Texas Red labeled cationized albumin with cell membranes of ECV 304 cells and an enhanced endocytic uptake compared to native albumin. The potential for introducing exogeneous DNA into cells was shown using NIH 3T3 fibroblasts. Successful, albeit low reporter gene expression could be achieved in the presence of chloroquine. Under in vitro conditions no toxic effect could be observed. In conclusion, cationized albumin may have promise as a non-toxic vector for gene delivery, especially for DNA vaccination.