Even if transfection efficiency of hippocampal neurons highly depends on cell culture preparation, transfection of primary neurons with Lipofectamine™ 2000 without Magnetofection leads to poor efficiency [3]. Instead, Magnetofection which associates magnetic beads as CombiMag reagent increases by 3.5 fold the transfection efficiency rate of lipofectamine 2000 (figure 1). Unfortunately, an important cellular toxicity reduced dramatically the number of living transfected cells after 5 days of incubation (Figure 1). This toxic effect was observed either in presence of CombiMag or in absence of CombiMag. It suggests that Lipofectamine 2000 reagent, DNA or GFP expression causes cell death. Also in most cells transfected with Lipofectamine 2000, segmentation or “clustering” of neurons appeared rapidly two or three days post transfection (Figure 2). Finally, neurons disappear from the culture dish and less healthy transfected neurons remain after several days of incubation. Transfections with PolyMag reagent which is a polymer based reagent associated with magnetic nanoparticles lead also to poor efficiency.
The best transfection efficiency was achieved by the use of NeuroMag which is a new magnetofection reagent developed by OZ Biosciences laboratories. This lipid based reagent is as efficient as Lipofectamine 2000 plus CombiMag but we did not observe any toxicity (Figure 1, 3). NeuroMag is a formulation and both the magnetic nanoparticles and the cationic lipid transfection reagent are combined yet. Also NeuroMag is ready to use and does not necessitate the use of other reagents in association with magnetic nanoparticles. In addition, nanoparticles are issued from a new generation of smaller magnetic beads with a diameter less than 200 nm which are more efficiently internalized by neurons.
In order to quantify more accurately the percentage of transfected neurons by using the NeuroMag reagent, neurons were stained with a MAP-2 antibody five days post transfection to discriminate neurons from glial cells (Figure 4). MAP2 is the major microtubule associated protein of brain tissue and is known to promote microtubule assembly and to form side-arms on microtubules. As it interacts with neurofilaments, actin, and other elements of the cytoskeleton, all neurons are stained by the MAP-2 antibody. We have observed by fluorescence microscopy that no glial cells were transfected by NeuroMag in the opposite of the use of the PolyMag reagent. Also we could determine that around 15% to 20% of the hippocampal neurons were transfected and express GFP. In comparison, other reagents give very low transfection efficiency.
Finally, to confirm that magnetofection technology does not perturb the localization of an active protein, we have transfected a plasmid DNA which encodes the recombinant NRB2 protein fused to GFP. NRB2 is a subunit of the NMDA neuron receptor witch is involved in the post synaptic transduction signal [4]. As expected the protein localizes all along the neurons as a punctuate labelling (Figure 5) which prove that nanoparticles does not inhibit the protein function in the cells.
In conclusion, NeuroMag allows to transfect efficiently primary neurons without observing any toxicity.
We are actually evaluating this technology in some in vivo applications [5]. The first results are promising but optimizations are still required. Magnetofection is not only limited to neurosciences applications but many publications relate its use for nucleic acid delivery inside a lot of primary cells as gastric cells or endothelial cells [6,7,8]. Also, this technology appears powerful to deliver a large range of molecules in primary cells and not only nucleic acids.
Acknowledgement
We thank Dr Igor Medina for providing us important transfection data.
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