Abstract
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.
Highlights
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
Transfection Efficiency Fluorescence Microscopy The uptake of plasmid DNA was primarily evaluated by comparing the GFP positive cells to the total quantity of cells showing blue DAPI fluorescence dye staining, attaining an estimate of the transfection efficiency
The transfection efficiency of cells transfected with the Plano-AuNP, Pulsed laser ablation in liquids (PLAL)-AuNP Size 1 and Size 2 protocol was apparently higher than that achieved with the conventional FHD transfection reagent or with the magnetic transfection protocols (MATra-A, MA Lipofection) (Images not shown)
Summary
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. Various methods and modifications are currently being evaluated to increase efficiency and reduce toxicity Both novel laser-based transfection methods [1] as well as nanoparticle (NP) approaches have been evaluated in recent studies [2,3,4]. Considering the latter, gold Nanoparticles (AuNPs) are in the focus of intense research due to their chemical stability, electrodensity and -affinity to biomolecules such as DNA, when these AuNPs are charged [5]. The inherent characteristics of the applied NPs could induce different toxic effects on cells due to several factors such as particle number and size, surface dose, surface coatings, degree of agglomeration, surface charges on particles and method of particle synthesis as well as post-synthetic modifications. The surface modification and surface charge can have a major impact on the biological response to various particles, the particle specific surface modification and the agents are an important factor that must be considered when choosing particular NPs [6]
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