Anti-EGFR-PEG-SPIO Nanomolecular Probe In MRgFUS Ablation Lung Adenocarcinoma Model Imaging, Sensitization And Efficacy Monitoring

Part ⅠPreparation of multifunctional anti-EGFR-PEG-SPIO nanoparticles molecular probe and its targeting MRI for H460 cellsObjective:To observe the trageting function of high affinity anti-EGFR monoclonal antibody(Cetuximab)-conjugated superparamagnetic iron oxide-dopamine(anti-EGFR-PEG-SPIO)lung cancer cells via epidermal growth factor receptor(EGFR)and the feasibility for surveillance of tumor targeting with MRI.Methods:An in vitro MRI was performed on that were incubated with anti-EGFR-PEG-SPIO and PEG-SPIO for 2 h.The cellular uptake of anti-EGFR-PEG-SPIO and PEG-SPIO was furtherevaluated by Prussian blue staining and transmission electron microscope(TEM).Nude mice of subcutaneously transplanted lung carcinoma were used as animal models,12 mice were divided into experimental group(n=6)and control group(n=6)randomly.Both were injected with anti-EGFR-PEG-SPIO and PEG-SPIO(1ml,40ug/mL)via caudal vein respectively.Tumor and muscle signal varying was observed after injection of contrast agent 0.5 hour,1 hour,2 hour,4 hour,6 hour,10 hour and 12 hour and CNR was calculated at different time points.Prussian blue staining and TEM of H460 cells and tumor was performed to observe intracellular irons.Results:The results of MTT assay,cell morphological observation,and hemolysis assay indicated that thetargeting anti-EGFR-PEG-SPIO nanoparticles show excellent and hemocompatibility and cytocompatibility.In vitro study,the targeting groupshowedthe signal intensity of H460 cells on T2WI decreased significantly compared with non-targeting group.In vivo MRI,there was a significant difference(F=89.02,P<0.01)of tumor signal to noise ratio(SNR)in targeting group and nontargeting group.There was statistic difference(F=31.18,P<0.01)of tumor SNR at different time before and after injection contrast agents in targeting group.There was statistic difference(F=4.32,P<0.01)of tumor SNR at different time before and after injection contrast agents in nontargeting group.The maximum intensify of the experiment group appears in 4-6 hours after injection and the control group appears in 0.5-2 hours after injection.Prussian blue staining and TEM results showed that a lot of intracellular irons were observed in H460 cells and tumor.Conclusion:The effect of active targeting via anti-EGFR in EGFR overexpressed cells could be achieved by anti-EGFR-PEG-SPIO in lung cancer cells in vitro and in vivo and this targeted delivery process could be monitored by MRI.Part ⅡActive targeting theranostic iron oxide nanoparticles for magnetic resonance-guided focused ultrasound ablation of lung cancerObjective:To study multi-function targeted anti-EGFR-PEG-SPIO nanoparticles probe for synergistic MRgFUS treatment of cancer and improve the tumor-ablative efficacy of a clinical MRgFUS system.Methods:Twenty-four tumor-burdened lung adenocarcinoma nude rats were randomly divided into 4 group:positive control group(high energy,74 W,n=6);negative control group(low energy,54 w,n=6);targeting anti-EGFR-PEG-SPIO group(32 w,n=6),non-targeting PEG-SPIO group(54 w,n=6).the peak temperatures of the four groups were real-time monitored by MRI sofrware and observed changed of peak temperatures in the tumor.Enhanced axial TiWI-weighted images after injection of gadolinium also analyzed after ablation.Results:During the ablation of MRgFUS,The sonication energy level used in the anti-EGFR-PEG-SPIO group(32 W)and PEGylated SPIO NPs group(54 W)were significantly lower than that in the positive control group(76 W)(P<0.01);In this study,the energy of the targeting group was even lower than that of the nontargeting group(32 W vs 54 W,P<0.01);in positive control group,anti-EGFR-PEG-SPIO group,PEG-SPIO group,a small black focal area of nonperfusion at series axial T1WI indicated the lack of blood supply in the tumor after treatment in targeting,nontargeting,and control group(high energy).Conclusion:This study showed that targeted anti-EGFR-PEG-SPIO nanoparticles probe,as synergistic agents,coulde significantly enhance the efficiency for in vivo ultrasonic energy deposition and reduce the injury of normal tissue surrounding tumor.Part Ⅲ Evaluation of tumor therapeutic efficacy after MRgFUS treatment of lung cancer using functional MR imaging(T2WI,DWI and DCE-MRI)Objective:To explore functional magnetic resonance imaging(T2WI,DWI and DCE-MRI)to evaluate tumor therapeutic efficacy after MRgFUS treatment of lung cancer.Methods:T2WI,DWI and DCE-MRI was performed at different time point(0.5 h,1,3,7,14,30 d)after MRgFUS ablation of lung cancer.T2 signal intensity and ADC values were measured in the around tumor and center of tumor.Ktrans,Kep and Vevalue was calculated at work station,the pearson test was used for correlation analysis.Results:There were statistical significance in T2 signal intensity and ADC value of targeting,non-targeting and positive control.ADC values in the tumor center significantly reduce after ablation(0.5 h),T2 signal intensity in the center of tumor significantly increase at different point after ablation.There were statistical significance before and after ablation(0.5 h)in T2 signal intensity and ADC values of targeting,non-targeting group(p<0.05).There were no statistical significance at different time point in T2 signal intensity of negative group(p>0.05).Axial contrast-enhance T1WI images after injection of Gd-DTPA showed a small focal area of nonperfusion in targeting,nontargeting,and control group(high energy),DCE-MRI show obvious enhance of tumor in control group(low energy).Ktrans Kep and Ve Values were statistically positively correlated with expression of EGFR(%)in four group(P<0.01).Conclusion:we demonstrated that a series of MR methods including T2-weighted image(T2WI),diffusion-weighted imaging(DWI)and DCE-MRI imaging could be utilized to noninvasively and conveniently monitor the therapeutic efficacy in rat models by MRgFUS.