Preparation And Experimental Study On The In Vitro Transfection Of PEI-PLL Magnetic Nanoparticles

Objective:To characterize superparamagnetic iron oxide nanoparticles modified PEI-PLL, and explore the cell transfection efficiency and its cell toxicity.Methods:1. Magnetic PEI-PLL was prepared by certain proportion oscillation mixture with superparamagnetism Fe3O4 nanoparticles and PEI-PLL after incubation, and the atomic arrangement, characterization of particle size, zeta potential etc were measured; pAd-Track plasmid was import in competent escherichia coli by chemical catalysis and then amplified and extracted it. The magnetic PEI-PLL combined with plasmid according to different proportions, and explored the best combination ratio by gel electrophoresis. Atomic force microscope was used to describe the atomic arrangement of composite product and zeta potential test the composite product.2.293 cells are grouped according to different transfection materials: 1) magnetic poly (ethylene imine + pAd - Track,2) non magnetic poly (ethylene imine + pAd - Track,3) GoldenTransfer + pAd - Track,4) PolyMAG + pAd - Track, each group separately set with and without magnetic field treatment on the control of the magnetic field, inverted fluorescence microscope in 24 h,48 h,72 h after transfection fluorescence expression in observation group cells, quantitative test of flow cytometry instrument transfection after 24 h of each carrier transfection rate;CCK8 kits each carrier effect on the activity of cell growth.Results:1. (1)According to the data of Sigma, magnetic iron oxide (II,III) is brown to dark brown powder, formula weight is 231.53g/mol and the particle size is 9-llnm (TEM), magnetization>45 emu/g; Atomic force microscope tells that an 18 carbon atoms of carbon chain was connected to magnetic iron oxide nanoparticles, and then modified with N-Hydroxysulfosuccinimide sodium salt(Sulfo-NHS). Sulfo-NHS on the magnetic Fe3O4 nanoparticles combined with PEI-PLL by amino, then formed Magnetic PEI-PLL. The diameter of magnetic product was 10-30nm, and the zeta potential is about+50mV. (2) Gel electrophoresis results showed that the more magnetic PEI-PLL combined more plasmid, and magnetic PEI-PLL can completely combine plasmid When the ratio of volume and mass reach 10:1. Atomic force microscope showed magnetic PEI-PLL connected with a number of five membered ring structure, which prove magnetic PEI-PLL could combine plasmid.2. (1) 24 hours after transfection, results of fluorescence microscope showed that transfection efficiency of magnetic or non-magnetic PEI-PLL had not much difference with GoldenTransferTM-D, while the expression of fluorescence was lower in PolyMAG; 48 hours later, the expression of fluorescence of magnetic and non-magnetic PEI-PLL increase markedly compared to GoldenTransferTM-D, and the increasing amount of PolyMAG is the lest; 72 hours after transfection, the expression of GFP were the highest, GoldenTransferTM-D took second place, and PolyMAG was the lowest; (2) Flow cytometry instrument detection showed that transfection efficiency of magnetic and non-magnetic PEI-PLL had no notable difference, and it was higher than other groups(P<0.05), transfection efficiency of GoldenTransferTM-D was a little higher than PolyMAG(P<0.05). In comparison with the results of magnetic field and non-magnetic field group, we found that magnetic field had no significant effect on the transfection efficiency(P>0.05); (3) Cell activity of magnetic and non-magnetic PEI-PLL group were affected less than other groups(P <0.05), and the cell activity were relatively higher at 48h after transfection, which is above 50% and there was no statistically significant differences(P >0.05). GoldenTransferTM-D and PolyMAG had visible effect on cell activity, and cell survival rate were about 10～20%. In comparison with the results of magnetic field and non-magnetic field group, we found that magnetic field had no significant effect on the cell activity.Conclusions:The preparation of magnetic PEI-PLL is successful, particle size is 10-30nm and uniform distribution. It carries positive charge and effectively combine plasmid. Transfection rate of magnetic PEI-PLL on 293 cell is higher, and it have low cytotoxicity.