Conjugation Of Iron Oxide Nanoparticles With Modified Folic Acid Or RGD For Targeted Molecular MR Imaging Of Non-small Cell Lung Cancer

PurposeTo study the synthesis,characterization,modification of iron oxide nanoparticles conjuated with folic acid or RGD and its application as molecular contrast agent in molecular MR imaging for non-small cell lung caner.Materials and methodsWater-dispersible Fe₃O₄ NPs synthesized via a mild reduction method wereconjugated with FA to generate FA-PEG-PEI-Fe₃O₄ NPs.USPIO NPs synthesized via a solvothermal route were conjugated with RGD peptide and modified with PEG to generate RGD-PEG-USPIO NPs.The final FA-PEG-PEI-Fe₃O₄ andRGD-PEG-USPIO NPs were characterized using various techniques such as X-ray diffraction?XRD?,¹H NMR,thermal gravimetric analysis?TGA?and transmission electron microscopy?TEM?.The cell cytotoxicity was evaluated by MTT assay.The specificity of FA-PEG-PEI-Fe₃O₄ NPs binding to FA receptor and RGD-PEG-USPIO NPs binding to integrin?_v?₃ was investigated in vitro by cellular uptake and cell MRI and in vivo by MRI of H460 tumors.Cellular uptake in vitro was confirmed byPrussian blue staining?ICP-OES and TEM analysis.Finally,the targeted Fe₃O₄ NPs were used in MR imaging of H460 and A549 lung cancer xenograft tumor model.The Prussian blue staining in vivo was performed to observe the Fe₃O₄ NPs in tumors.The biodistribution of the iron materials was quantified studied by ICP-OES system.Results?1?FA-PEG-PEI-Fe₃O₄ NPs synthesized via a mild reduction method were quite uniform,stable,and have a core diameter of 8.9 nm.The NPs have stronger T₂negative enhancement effect.The T₂ relaxation rate of the NPs is as high as 440.01mM^-1S^-1,and can be used as a molecular imaging of T2 contrast agents.MR cell imaging results show that with the increase of Fe concentration,MRI signal of the cells reduces gradually.The signal intensity from the cells treated with FA-PEG-PEI-Fe₃O₄ NPs is clearly lower than that of similar cells treated with FA blocking at the same Fe concentration.The tumor MR signal from the mice injected with FA-PEG-PEI-Fe₃O₄ NPs decreased from 0.35 h to 0.8 h compared with the signal obtained for mice that were blocked with FA.0.8 h after injection,the MR signal of the two tumors was recovered.In vivo tissue distribution shows that the FA-PEG-PEI-Fe₃O₄ NPs have high biological safety.Also the results of cell viability assay and cell morphological observation indicated that the targeted NPs exhibit excellent cytocompatibility and biocompatibility.?2?RGD-PEG-USPIO NPs were prepared by a solvothermal route had a core diameter of 2.68 nm and TEM observation showed no obvious cluster particles.The hydrodynamic size of the USPIO NPs was 13.8nm.Its T₁ relaxation rate reached 1.41mM^-1S^-1.Cell Prussian blue staining,ICP-OES quantitative analysis and TEM microscope all confirmed that the targeted NPs had good specific binding ability to A549 cells.The signal intensity from the A549 cells treated with RGD-PEG-USPIO NPs is clearly higher than that of the same cells treated with PEG-USPIO NPs at the same iron concentration.The tumor MR signal from the mice injected with RGD-PEG-USPIO and PEG-USPIO NPs increased after the injection of 0.5,1.0 and1.5 h,and the tumor treated with RGD-PEG-USPIO NPs increased obviously than that of tumor treated with PEG-USPIO NPs.The MR signal decreased gradually after1.5 h injection and the signal strength of two groups returned to normal at 3 h after injection.Conclusion?1?The prepared FA-PEG-PEI-Fe₃O₄ NPs have a specific binding ability to H460cells with FR over-expression and can offer significant potential for use as contrast agents for targeted T₂ MR imaging of H460 lung cancer tumors.?2?The developed RGD-PEG-USPIO NPs via a solvothermal method have stronger T₁ paramagnetic effect at high concentrations.The RGD-PEG-USPIO NPs show significant potential for use as T₁ contrast agents in targeted molecular probes for A549 tumor-bearing mice.