The Effect Of Magnetic Field Of Different Uniformity On Magnetofection Efficiency

Objective:To investigate the effect of uniform magnetic field on magnetofection and provide a new method to improve the transfection rate.Methods:1.Polyethyleneimine（PEI）modified ferroferric oxide（Fe₃O₄）nanoparticles（PEI-Fe₃O₄）werepreparedbychemical coprecipitation and the morphology,composition,the pDNA binding ability were charaterized using transmission electron microscope（TEM）,laser diffraction particle size analyzer,vibrating sample magnetometer（VSM）,atomic force microscopy and agarose gel electrophoresis.A zeta potential analyzer was used to measure the electric potential.2.The uniformity of the unilateral Halbach magnet and the 96 holes magnetweremeasuredbyatestingplatformconsistingof three-dimensional motion platforms and gaussian meters.3.（1）The expression efficiency GFP in both HEK-293 and MG-63 cell lines under different uniformity of magnetic field was detected with inverted fluorescence microscope and flow cytometry.（2）Distribution of transfection under different magnetic fields was obsesrved by fluorescence imaing system.4.（1）The cell viability of PEI-Fe₃O₄ in HEK-293 and in MG-63 were studied by MTT method;（2）TEM was used to observe the phagocytosis progress of PEI-Fe₃O₄ nanoparticles.Results:1.（1）PEI-Fe₃O₄ nanoparticles were well prepared;The Fe₃O₄nanoparticles observed through TEM were circular or oval and had favorable evenness.Fe₃O₄ nanoparticles’average size was 7.292nm,the electric potential of PEI-Fe₃O₄ nanoparticles was positive and the zeta potential was 36.8±0.5 mV.The potential of PEI-Fe₃O₄-pDNA was reduced but still positive.The hysteresis loop of PEI-Fe₃O₄ nanoparticles showed that a small remnant magnetization of 0.0177emu/g was obtained at the external magnetic field of 0 Oe,indicating that the magnetic particles produced were superparamagnetic.（2）The results of agarose gel electrophoresis（AGE）analysis and identification showed that the superparamagnetic PEI-Fe₃O₄ has a good compound ability for plasmid pDNA,and its optimal combination ratio is N/P=0.5;The Atomic Force Microscope（AFM）showed that the morphology was in line with the expected chemical structure diagram.2.The results showed that the difference of uniformity of the two magnetic fields was about 100 times.In this case,we set the 96-hole magnetic plate as the Nonuniform MF and the Halbach magnetic field is a relative Uniform MF.3.（1）The inverted fluorescence microscope showed that in HEK-293and MG-63 cell line:no matter the presence of magnetic field,compared with the naked plasmid group,the PEI transfection group were higher;There is little difference between PEI-pDNA and PEI-Fe₃O₄-pDNA without MF group;PEI-Fe₃O₄-pDNA+Nuniform MF is significantly higher than than that of PEI-Fe₃O₄-pDNA+Nonuniform MF.（2）Flow cytometry showed that the transfection efficiency in HEK-293 was:PEI-Fe₃O₄-pDNA+Uniform MF（77.75±0.07%）>PEI-Fe₃O₄-pDNA+Nonuniform MF（30.65±0.49%）>PEI-Fe₃O₄-pDNA（7.90±0.56%）（p<0.05）;The transfection efficiency in MG-63 was PEI-Fe₃O₄-pDNA+Uniform MF（10.65±0.21%）>PEI-Fe₃O₄-pDNA+Nonuniform MF（5.60±0.28%）>PEI-Fe₃O₄-p DNA（2.40±0.14%）（p<0.05）;（3）The results of fluorescence imaing system show that the distribution of magnetic transfection is more uniform than that of the non-uniform magnetic field.4.（1）MTT showed that the cell survival rate in PEI-Fe₃O₄-pDNA+Uniform MF and PEI-Fe₃O₄-pDNA+Nonuniform MF group were reduced compared with the control（p<0.05）,but the PEI-Fe₃O₄-pDNA+Uniform MF and PEI-Fe₃O₄-pDNA+Nonuniform MF group had no statistical difference between the cell survival rate（p>0.05）,and were higher than PEI-Fe₃O₄-p DNA and PEI-pDNA（p<0.05）in both HEK-293and MG-63 cell lines;（2）PEI-Fe₃O₄ magnetic nanoparticles can be effectively consumed by cells and have little effect on cell morphology.Conclusions:In this paper,PEI-Fe₃O₄-pDNA nanoparticles was successfully prepared,and the uniformity of the two magnetic fields was determined,and a new type of magnetic transfection system was constructed.When the MF’s intensity is unaltered,the magnetic transfection efficiency inceases by the incease of MF’s uniformity,and the combination of the unilateral Halbach magnet and the magnetofection can be used as a new method to improve the transfection rate which may potentially be employed in gene therapy.