Tansfection of ovarian cancer SKOV3 cells with SPIO-PLL-pshRNA molecular probes and MR imaging

Abstract:

Objective：To prepare SPIO-PLL-pshRNA molecular probes and to preliminarily evaluate the feasibility of transfecting SKOV3 cells and MR imaging in vitro. Methods：Superparamagnetic iron oxide modified with Poly-L-lysine（SPIO-PLL） was bound with plasmid pGenesil1-shRNA through electrostatic absorption. Concentration of plasmid DNA in the supernatant was detected by micro-ultraviolet spectrophotometer after the centrifugation of the complexes. By this way，the ability of plasmid DNA binding to SPIO-PLL could be evaluated. Conjunction between SPIO-PLL and plasmid DNA was confirmed via gel retardation experiments and zeta potential methods. Cell transfection was evaluated with Prussia blue staining for iron assessment and fluorescence microscope for green fluorescent protein（GFP） expression detection. Changes of cells in MR signal intensities were observed after the transfection. Results：DNA binding tests，gel retardation experiments and zeta potential methods demonstrated that SPIO-PLL could effectively bind plasmid DNA when the w/w ratio of SPIO-PLL and plasmid DNA was more than 6∶1. zeta electric potentials of SPIO，SPIO-PLL and SPIO-PLL-pshRNA were （-14.8±0.35），（15.2±0.55） mV and （3.6±0.35） mV，with statistical differences among groups（F=6323.782，P=0.000；LSD：all P=0.000）. After transfection with the SPIO-PLL-pshRNA complexes，iron particles could be detected in cells with Prussia blue staining；green fluorescence could be observed by fluorescent microscope；MR imaging showed T2*WI signal intensities reduction（558±19，427±16，283±19 and 191±8 in non transfection group，10，20 μg/ml and 40 μg/ml groups respectively；600±36 and 670±16 in agarose group and distilled water group）. The signal intensity was decreased with the increase of complex concentration and statistical differences were observed among groups（F=279.667，P=0.000；SNK-q：all P<0.05）. Conclusions：SPIO-PLL nanoparticles can effectively bind with plasmid pGenesil1-shRNA as SPIO-PLL-pshRNA molecular probe. This probe can successfully transfect SKOV3 cells and T2*WI signal intensity reduction can be observed by MR scanning.