Nanomagnetic Gene Transfection for Non-Viral Gene Delivery in NIH 3T3 Mouse Embryonic Fibroblasts

The objective of this work was to examine the potential of oscillating nanomagnetic gene transfection systems (magnefect-nano™) for improving the transfection efficiency of NIH3T3 mouse embryonic fibroblasts (MEFs) in comparison to other non-viral transfection techniques—static magnetofection™ and the cationic lipid agent, Lipofectamine 2000™. Magnetic nanoparticles (MNPs) associated with the plasmid coding for green fluorescent protein (GFP) were used to transfect NIH3T3 cells. The magnefect-nano system was evaluated for transfection efficiency, and any potential associated effects on cell viability were investigated. MNPs associated with the plasmid coding for GFP were efficiently delivered into NIH3T3 cells, and the magnefect-nano system significantly enhanced overall transfection efficiency in comparison to lipid-mediated gene delivery. MNP dosage used in this work was not found to affect the cell viability and/or morphology of the cells. Non-viral transfection using MNPs and the magnefect-nano system can be used to transfect NIH3T3 cells and direct reporter gene delivery, highlighting the wide potential of nanomagnetic gene transfection in gene therapy.


Introduction
In recent years, conventional therapies have provided limited improvement in treating complex diseases, creating an increasing need for the investigation of gene therapy as a potential tool for clinical and/or scientific research purposes [1][2][3].
Despite the success of viral vectors at transfecting a variety of primary cells and cell lines, over the past decade, there has been an increasing shift towards the use of non-viral gene transfection due to viral-associated concerns, mostly regarding safety, such as induced inflammatory response and mutagenesis, as well as limitations on the plasmid size [4][5][6][7][8].
As gene therapy and stem cell therapy are closely related, very often using embryonic stem cells and stem cell lines for their clinical and research applications, MEFs have demonstrated a key role for the expansion and maintenance of embryonic stem cells as their feeder layers [9], as well as their different applications in tissue engineering [10,11]. Up to now, limited work has been done to investigate the potential of transfecting MEFs (NIH3T3 cells) for their use in tissue engineering and stem cell biology.
Here, we investigate the potential of the novel non-viral, nanomagnetic gene transfection in the presence of oscillating magnetic fields for NIH3T3 transfection.
Originally, magnetic nanoparticle-based gene transfection was demonstrated by Mah, Byrne et al. more than a decade ago [12][13][14][15]. The group used magnetic microspheres with GFP-carrying rAAV linked to the microspheres via heparin sulfate. The complex was magnetically targeted to a specific region of a culture of HeLa cells. The magnetic targeting enabled highly efficient uptake of the GFP gene into HeLa cells localized at the site of the applied magnetic field.
In further experiments, Plank, Scherer, Rosenecker and others developed magnetic nanoparticles for non-viral "magnetofection" use, in which plasmid DNA or siRNA is coupled directly to magnetic nanoparticles coated with charged polymers to which the plasmids adhere [14][15][16][17]. High-field, high-gradient permanent magnets placed beneath the culture plate rapidly draw the particle/DNA complex into contact with the cells in culture, where it is taken up via endocytosis.
In 2003, in order to improve the transfection efficiency of magnetofection-type transfection techniques, our group began developing a variation of magnetofection, which employs oscillating magnet arrays to promote more efficient transfection via mechanical stimulation of endocytosis. The technique generally improves transfection efficiency, but also maintains the advantages of magnetofection-rapid transfection and high cell viability in comparison to other non-viral transfection methods [18][19][20]. In addition, studies by our group and others on magnetic nanoparticles for regenerative medicine and gene transfection have shown that these techniques have little or no toxic effects on cells [19][20][21][22][23][24].
The technique works by attaching DNA or siRNA to magnetic nanoparticles coated with a charged polymer, which condenses the DNA on the surface. The complex is placed into cell culture plates and DNA coding for GFP) (Plasmid Giga Kit, QiaGen, West Sussex, UK) in Knock-out DMEM media (Gibco-Invitrogen, UK) using spectrophotometry. nTMag MNPs are biocompatible and biodegradable, 100 nm in diameter, superparamagnetic nanoparticles (when used as per manufacturer's instructions) with magnetite cores coated with proprietary multilayer PEI derivate. nTMag MNPs have been provided with accompanying quality control information that includes pH value of suspension (7.0), particle size distribution (1.7) and zetapotential (+23.87 mV). A range of different nTMag concentrations were mixed with a fixed volume of pEGFP-N1 plasmid (30 μL). The proportion of DNA that remained unbound (i.e., still in solution after centrifugation) was then determined. A sample containing only DNA and Knock-out DMEM media, but no nTMag MNPs, was assayed as a control. Following mixing of the MNPs and DNA at room temperature (RT) for 15 min, samples were centrifuged at 14,000 rpm for 5 min to induce sedimentation of the particles and/or particle:DNA complexes. The absorbance of the supernatant containing the unbound DNA was measured for each sample at 260 nm and compared with the Knock-out DMEM blank sample. In order to determine the proportion of unbound (free) DNA, absorbance readings were expressed as percentages of the absorbance of the DNA-only (blank) control.

Transfection of NIH3T3 Cells Using the Magnefect-Nano System
Experimental evaluation of transfection efficiency was performed using transfection complexes composed of 100 μL of Knock-out DMEM medium, 0.2 μg DNA and 0.2 μL nTMag MNPs per 96 well tissue culture plate well (Sigma-Aldrich, Dorset, UK). Following the addition of the particle/DNA complexes to the cell cultures, samples were transferred to an incubator at 37 °C, 5% CO 2 and placed over the magnefect-nano oscillating magnet array (nanoTherics Ltd., Stoke-on-Trent, UK) for 30 min. At 30 min post-transfection, the cell culture plates were removed from the magnefect-nano system, and transfection complexes were replaced with 100 μL of supplemented medium (as described previously). All samples were transferred back into an incubator for 48 h before analysis.

Transfection of NIH3T3 Cells with Lipofectamine 2000
NIH3T3 cells were maintained as described previously and seeded onto 96 well tissue culture plates. Lipofectamine 2000 complexes were composed of 100 μL Knock-out DMEM medium, 0.2 μg pEGFP-N1 DNA and 0.5 μL Lipofectamine 2000 per 96 well tissue culture plate well, as per the manufacturer's recommended protocol. Following the addition of complexes, samples were transferred into an incubator at 37 °C, 5% CO 2 , and complexes were left on the cell cultures for 6 h, as per the manufacturer's protocol. At 6 h post transfection, all transfection complexes were replaced with 100 μL supplemented medium (as described previously). All samples were transferred back into an incubator for 48h before analysis.

Immunofluorescence & Fluorescence Activated Cell Sorting (FACS) Assays
Following 48 h of incubation, immunofluorescent microscopy was performed to acquire fluorescent images of NIH3T3 cells labeled with Phalloidin-TRICH actin stain (Sigma-Aldrich, Dorset, UK) and GFP from GFP-expressing transfected NIH3T3 cells, as shown in Figure 3. Images were captured with  great potential for use as an effective, non-viral transfection agent for MEFS. A major advantage of this technique for use in regenerative medicine and tissue engineering research applications is the fact that it is both non-viral and does not impact cell viability, though a thorough investigation of up-or down-regulation of off-target genes will be needed before the technique could be used in a clinical setting.