Preparation And Preliminary In Vitro Study Of Ovarian Cancer Specific Molecular Probe

Objective: To prepare SPIO-PLL-pshRNA molecular probe, and thenpreliminarily evaluate the feasibility of which transfecting SKOV3cells andMR imaging in vitro.Methods: SPIO-PLL binded with plasmid pGenesil1-shRNA throughelectrostatic absorption. Concentrations of pasmid DNA in the supernatewere detected by Micro-Ultraviolet Spectrophotometer after centrifugationof the SPIO-PLL-pshRNA complexes at different weight rations. By thisway, the ability of plasmid DNA binding to SPIO-PLL could beevaluated,and the optimal weight proportion of preparing probes could bedetermined. Conjunction between SPIO-PLL and plasmid DNA wasconfirmed via gel retardation experiments and zeta potential detection. Thecytotoxicity of probes was explored with CCK-8method. The cellularuptake of probes was preliminarily evaluated with Prussia blue staining foriron assessment and further assessed with Fluorescence microscope forGFP expression detecting. The intracellular distribution of iron particalscontained in probes was observed by transmission electron microscope.Thechanges of MR signal intensities in transfected cells were detected throughr MR scanning.Result: DNA binding tests and gel retardation experiments resultedthat SPIO-PLL could effectively bind plasmid DNA when the weight ratioof SPIO-PLL and plasmid DNA reached6:1.The zeta potential values ofthree sample groups(including SPIO,SPIO-PLL and SPIO-PLL-pshRNA)were respectively (-14.8±0.35) mV,(15.2±0.55) mV and (3.6±0.35)mV,which showed statistically significant differences amongthem(（ANOVA：F=6323.782，P=0.000；LSD：all P=0.000）). The resultof cytotoxicity test indicated that the cell survival rate was higher than80%when the concentration was less than50mg/L. After transfection with theprobes, iron particals could be detected in cells with Prussia bluestaining,green fluorescene could be observed through fluorescentmicroscope,and the iron particals located in the cytoplasm observed undertransmission electron microscope. MR imaging showed signal intensitiesreduction after cells transfected with probes on T2*-weighted imaging. Andthe higher concentration of probe was, the lower signal intensity showed.There was statistically significant difference in T2*value among differentconcentration groups（ANOVA：F=279.667，P=0.000；SNK：all P<0.05）.Conclusion:An ovarian cancer specific molecular probe can besuccessfully prepared, which can succeed in transfecting ovarian cancerSKOV3cells with low cellular toxicity, and be sensitively detected by MR scanning with T2*-weighted sequence.