Comparison of nanoparticle-mediated transfection methods for DNA expression plasmids: efficiency and cytotoxicity

Journal of Nanobiotechnology

Research

Karsten Feige¹  María Carolina Durán¹  Jessika-M V Müller¹  Stephan Barcikowski²  Jan T Soller³  Annette Barchanski⁴  Hugo Murua Escobar⁵  Ingo Nolte⁵  Saskia Willenbrock⁵  Arianna Maiolini⁵ 

[1] Equine Clinic, University of Veterinary Medicine Hannover, Buenteweg 9, 30559, Hannover, Germany;Institute of Technical Chemistry I, University of Duisburg-Essen and Center for Nanointegration Duisburg-Essen (CeNIDE), Universitaetsstr. 5-7, 45141, Essen, Germany;Institute of Veterinary Medicine, University of Goettingen, Burckhardtweg 2, 37077, Goettingen, Germany;Laser Zentrum Hannover e.V., Hollerithallee 8, 30419, Hannover, Germany;Small Animal Clinic and Research Cluster of Excellence "REBIRTH", University of Veterinary Medicine, Buenteweg 9, 30559, Hannover, Germany;

关键词: Propidium Iodide;    Transfection Efficiency;    High Transfection Efficiency;    Transfection Protocol;    Cell Proliferation ELISA;   

DOI  :  10.1186/1477-3155-9-47

 received in 2011-06-21, accepted in 2011-10-20,  发布年份 2011

来源: Springer

【 摘 要 】

BackgroundReproducibly high transfection rates with low methodology-induced cytotoxic side effects are essential to attain the required effect on targeted cells when exogenous DNA is transfected. Different approaches and modifications such as the use of nanoparticles (NPs) are being evaluated to increase transfection efficiencies. Several studies have focused on the attained transfection efficiency after NP-mediated approaches. However, data comparing toxicity of these novel approaches with conventional methods is still rare.Transfection efficiency and methodology-induced cytotoxicity were analysed after transfection with different NP-mediated and conventional approaches. Two eukaryotic DNA-expression-plasmids were used to transfect the mammalian cell line MTH53A applying six different transfection protocols: conventional transfection reagent (FuGENE HD, FHD), FHD in combination with two different sizes of stabilizer-free laser-generated AuNPs (PLAL-AuNPs_S1,_S2), FHD and commercially available AuNPs (Plano-AuNP), and two magnetic transfection protocols. 24 h post transfection efficiency of each protocol was analysed using fluorescence microscopy and GFP-based flow cytometry. Toxicity was assessed measuring cell proliferation and percentage of propidium iodide (PI%) positive cells. Expression of the respective recombinant proteins was evaluated by immunofluorescence.ResultsThe addition of AuNPs to the transfection protocols significantly increased transfection efficiency in the pIRES-hrGFPII-eIL-12 transfections (FHD: 16%; AuNPs mean: 28%), whereas the magnet-assisted protocols did not increase efficiency. Ligand-free PLAL-AuNPs had no significant cytotoxic effect, while the ligand-stabilized Plano-AuNPs induced a significant increase in the PI% and lower cell proliferation. For pIRES-hrGFPII-rHMGB1 transfections significantly higher transfection efficiency was observed with PLAL-AuNPs (FHD: 31%; PLAL-AuNPs_S1: 46%; PLAL-AuNPs_S2: 50%), while the magnet-assisted transfection led to significantly lower efficiencies than the FHD protocol. With PLAL-AuNPs_S1 and _S2 the PI% was significantly higher, yet no consistent effect of these NPs on cell proliferation was observed. The magnet-assisted protocols were least effective, but did result in the lowest cytotoxic effect.ConclusionsThis study demonstrated that transfection efficiency of DNA-expression-plasmids was significantly improved by the addition of AuNPs. In some combinations the respective cytotoxicity was increased depending on the type of the applied AuNPs and the transfected DNA construct. Consequently, our results indicate that for routine use of these AuNPs the specific nanoparticle formulation and DNA construct combination has to be considered.

【 授权许可】

Unknown   
© Durán et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.