Optimization of nonviral transfection: variables influencing liposome-mediated gene transfer in proliferating vs. quiescent cells in culture and in vivo using a porcine restenosis model

Abstract

Cationic liposomes/DNA complexes are widely used vectors for delivering genes in clinical and experimental trials. Relatively low transfer efficiencies in vivo compared with viral gene transfer may be improved using local application. In addition, markedly increased transfer efficiency may be achieved in vitro and in vivo via optimization of known variables influencing liposomal transfection. Lipofection under different conditions was performed in various cell lines and primary porcine smooth muscle cells. Optimized conditions found in vitro were verified in vivo using a porcine restenosis model. Toxicity was monitored analyzing cell metabolism. Transfer efficiency and safety were determined using morphometry, histology, galactosidase assays, PCR, and RT-PCR. The most important variables enabling maximum transfer efficiency were firstly the appropriate selection of cationic lipids for the cell type to be transfected, secondly the DNA/liposome ratio chosen, which depended on the cell type and cationic lipids used, and thirdly the state of proliferation of the targeted cells. Transfection in vivo demonstrated two- to fivefold higher transfer efficiencies when transfer conditions were extrapolated from optimization experiments in stationary cells compared with the use of conditions established in proliferating cells. Application of the therapeutic gene for cecropin using optimized transfer conditions resulted in a significantly reduced neointima formation compared with the transfection using a control gene for ss-galactosidase. Thus, in this vascular model, initial optimization of lipofection in stationary cells in culture followed by local delivery in vivo and with selection of a suitable therapeutic gene led to markedly improved transfer efficiencies, gene expression, and biological effect. Stationary cell cultures simulate more realistically the in vivo situation and may therefore represent a better model for future in vivo experiments. In addition, the advantages of liposomes are easy handling, low toxicity, and the lack of carcinogenicity or immunogenic reactions.

Transfection of myeloid leukaemia cell lines is distinctively regulated by fibronectin substratum

“…The liposome-mediated transfection interferes with the cell viability. The toxicity arisen from the cationic lipid-DNA complex may vary according to chemical composition of the liposome, the amount of DNA, and the type of the host cell (Uchida et al 2002;Pelisek et al 2002;Nguyen et al 2007). Therefore, the efficiency of gene delivery is generally inversely proportional to the cell viability.…”

“…Proliferating cells are better targets for liposomal gene transfer (Mortimer et al 1999;Pelisek et al 2002). In contrast, actively dividing leukaemia cells grown in suspension exhibit a low transfection capacity that indicates other possible factors influencing the efficiency of gene transfer (Uchida et al 2002).…”

Small Poly-<i>L</i>-Lysines Improve Cationic Lipid-Mediated Gene Transfer in Vascular Cells in vitro and in vivo

“…As a control, GT with lipoplexes of DOCSPER without sPLLs was performed. The DOCSPER/DNA ratio used for proliferating cells was 6/1 and for stationary SMCs 10/1 [14, 22]. In proliferating SMCs, lipopolyplexes containing L18 displayed an up to 2-fold higher transfection efficiency than the corresponding lipoplexes.…”

“…For the inhibition of SMC proliferation, cells were incubated with 300 µg/ml heparin (Braun, Melsungen, Germany) for further 24 h before transfection and following each medium change [14]. GT by using the cationic lipid DOCSPER was performed as described previously [14, 22]. Four hours following complex addition, transfection medium was replaced with fresh growth medium, and after an additional 2 days, GT efficiency was determined by measuring β-galactosidase activity.…”

“…For flow cytometric analysis, proliferating SMCs were seeded into 12-well plates and cultivated as previously described [9, 14, 22]. Transfection with Cy5-labelled (excitation at 650 nm, emission at 670 nm) plasmid DNA (Mirus Bio Corporation, Madison, Wisc., USA), unlabelled sPLLs and DOCSPER was done as described above.…”