MRI And PET Molecular Imaging Experimental Study For The Tumor Angiogenesis Of Nasopharyngeal Carcinoma

PartⅠ The synthesis and evaluation of ultra small superparamagnetic iron oxide nanoparticles conjugated with RGD peptide RGD-PAA-USPIOPurpose: Arginine-glycine-aspartic acid(RGD)peptides can specifically target integrin avβ3.The aim of this study was to synthsize ultra small superparamagnetic iron oxide(USPIO)nanoparticles conjugated with RGD peptides RGD-PAA-USPIO nanoparticles and to validate its capability for targeting integrin αvβ3 expressed on human umbilical vessel endothelium cells(HUVECs)in vitro.Materials and methods: The USPIOs were coated with polyacrylic acid(PAA)by polyol method and then conjugated with RGD peptides,forming the RGD-PAA-USPIO nanoparticles.Transmission electron microscope(TEM)and Dynamic light scatting(DLS)were used to measure the core diameter and hydrodynamic diameter of PAA-USPIO and RGD-PAA-USPIO nanoparticles.The longitudinal(r1)and transverse relaxation(r2)of RGD-PAA-USPIO nanoparticles was also determined in vitro.Meanwhile,HUVECs were cultivated with PAA-USPIO、 RGD-PAA-USPIO and RGD-PAA-USPIO+RGD for different time and then underwent Prussian blue staining and inductively coupled plasma mass spectrometer(ICP-MS)and cell MR imaging studies to determine its specific targeting capability.In addition,TEM was also performed to explicit the subcellular localization of RGD-PAA-USPIO nanoparticles.Results: The core diameter of PAA-USPIO and RGD-PAA-USPIO was about 14.5nm,while the hydrodynamic diameter of PAA-USPIO and RGD-PAA-USPIO was 49.23 nm and 59.44 nm,respectively.RGD-PAA-USPIO nanoparticles presented with rounded shape and homogeneous distribution pattern on TEM.The r1 and r2 values of RGD-PAA-USPIO nanoparticles were 8.7m MS-1 and 137.85 m MS-1,respectively.The RGD-PAA-USPIO nanoparticles have obvious effect on the attenuation of T2 relaxation,owing to the ratio of r2 and r1 as high as 15.84.Prussian blue staining demonstrated that the positive staining areas of iron were more extensive with incubation time extended,especially for the incubation of RGD-PAA-USPIO with HUVECs,compared with the control group(PAA-USPIO)and the competition group(RGD-PAA-USPIO +RGD).The mean value of cellular iron content within HUVECs incubated with RGD-PAA-USPIO at the time point of 30 min,1hour,and 3 hours was 0.427±0.021 pg/cell,0.587±0.014 pg/cell and 0.743±0.021 pg/cell,indicating time-dependence trend.In the same way,the mean T2 value of HUVECs after incubation with RGD-PAA-USPIO for different time as above were 113.33±5.03 ms,102.67±14.57 ms and 82.33±3.51 ms,respectively.TEM examination gave the subcellular localization of RGD-PAA-USPIO,demonstrating that large amounts of RGD-PAA-USPIO nanoparticles were internalized and accumulated in the cellular endosomes by specifically mediated by the interaction between the RGD peptides and integrin αvβ3.Conclusion: RGD-PAA-USPIO nanoparticles have the capability to target the integrin αvβ3 expressed on HUVECs in vitro,laid a foundation for in vivo studies.Part Ⅱ The in vivo study of molecular probe RGD-PAA-USPIO for tumor angiogenesis imaging in Human Nasopharyngeal Carcinoma Xenograft ModelPurpose: To investigate the biodistribution and the targeted imaging capability of molecular probe RGD-PAA-USPIO in human nasopharyngeal carcinoma(NPC)xenograft model.Materials and Methods: The expression of integrin αvβ3 on the surface of CNE-2 cells line was confirmed by Western blot and flow cytometry and immunofluorescence staining,with HUVECs as the positive control.Mice bearing human NPC tumor xenografts underwent MRI scanning with T2 WI and T2 mapping sequences on different time points before the injection of nanoparticles and 1h、2h、4h、6h、8h、12h and 24 h after receiving the same dose of RGD-PAA-USPIO and PAA-USPIO via tail vein.T2 values of different organs were measured at each time point,confirmed by Prussian blue staining.For the tumor tissues,the best imaging time point was obtained from the time-intensity curve of the tumors.Mice bearing human NPC tumor xenografts also underwent MRI scanning on the best imaging time point after receiving the same dose of RGD-PAA-USPIO and PAA-USPIO and RGD-PAA-USPIO+RGD via tail vein injection,and then ΔT2 values of the tumors were calculated.Results: The integrin αvβ3 expression on the CNE-2 cell was negligible,confirmed by Western blot and flow cytometry and immunofluorescence staining.The T2 value in the liver of mice bearing human NPC tumor xenografts decreased significantly after receiving the same dose of RGD-PAA-USPIO and PAA-USPIO,while these values in the muscle and kidney changed a little.Prussian blue staining showed that the iron particles were mainly distributed in the liver and spleen,and no obvious iron particles were observed in kidney and muscle.The time-signal curve of tumors showed that 6 hours after injection of RGD-PAA-USPIO was the best imaging time for evaluating tumor angiogenesis.In addition,the ΔT2 values of the tumors were significantly higher than those in the control and competitive groups(P<0.01).The specific binding of RGD-PAA-USPIO on the surface of the tumor angiogenesis was confirmed by Prussian blue staining.Conclusion: MR molecular probe RGD-PAA-USPIO was mainly distributed in the reticuloendothelial system such as liver and spleen.No significant distribution was observed in kidney and muscle.Moreover,RGD-PAA-USPIOs are capable of noninvasive detecting tumor angiogenesis in human NPC xenograft model.Part Ⅲ Noninvasive monitoring of early antiangiogenic therapy response using RGD-conjugated ultrasmall superparamagnetic iron oxide nanoparticles in Human Nasopharyngeal Carcinoma Xenograft ModelPurpose: To evaluate the potential role of a novel arginine-glycine-aspartic acid(RGD)-coupled,polyacrylic acid(PAA)-coated ultrasmall superparamagnetic iron oxide(USPIO)(referred to as RGD-PAA-USPIO)in monitoring the early response to antiangiogenic treatment in human nasopharyngeal carcinoma(NPC)xenograft model.Materials and Methods: The study was approved by the institutional animal care and use committee.Mice bearing human NPC tumor xenografts were intraperitoneally injected with Endostar(n=10)or normal saline(n=10)at the same dose of 20mg/kg/d for consecutive 14 days.Magnetic resonance imaging with T2 WI and T2 mapping sequence was performed before and after injection of RGD-PAA-USPIO at the time point of 6 hours.ROIs were manually drawn on the tumor tissue to avoid cyst and necrosis and hemorrhage areas,and then ΔT2 and ΔR2 values were calculated.For the assessment of antiangiogenesis therapy,MRI examination were performed baseline and after 4 and 14 days of Endostar or normal saline treatment.Tumors were harvested at the end of imaging time points for histopathological analysis with H&E and microvessel density(MVD)and integrin avβ3 immunostaining.All data was expressed with mean ± standard deviation.Statistical analysis was performed by using the Student’s t test or Mann-Whitney U test with Graph Pad Prism 5.0(Graph Pad Software Inc.,La Jolla,CA).P value < 0.05 was considered statistically significant.Results: A much less time-dependent increase in tumor growth was observed in the treated group than the control group after consecutively antiangiogenic therapy with Endostar for 14 days,and a significant difference in tumor volume was observed between the two groups from day8 after therapy onset(P < 0.05).Endostar treatment resulted in a significant reduction of the R2 changes compared with the control group both at day4 and day14,confirmed by the immunohistochemistry of MVD after treatment.The ΔT2 values of tumor in the treatment group on day4 and day 14 after therapy onset were 21.3±3.27 ms and 10.4±2.50 ms,while the ΔR2 values of the tumors were 1.82±0.32 s-1 and 0.83±0.24s-1,respectively.Both the ΔT2 and ΔR2 values were significantly lower than those in the control group(P<0.01).In addition,there is statistical significance among the different time points of the ΔT2 and ΔR2 values of the tumors in the treatment group,confirmed by the immunofluorescence of CD31 and integrin β3 after treatment.Conclusion: This study demonstrates that RGD-coupled,PAA-coated USPIOs nanoprobes,RGD-PAA-USPIOs,are capable of noninvasive monitoring of the tumor response to Endostar antiangiogenic therapy in human NPC xenograft model at early stages of treatment.Part ⅣThe Feasibility of 18F-Al F-NOTA-PRGD2 PET/CT for Monitoring Early Response of Endostar Antiangiogenic Therapy in Human Nasopharyngeal Carcinoma Xenograft Model compared with 18F-FDGPurpose: Radiolabeled arginine-glycine-aspartic acid(RGD)peptides allow specifically targeting integrin avβ3,a protein overexpressed during angiogenesis,leading to great potential for noninvasively evaluating the status of tumor angiogenesis and monitoring antiangiogenic treatment.The aim of this study was to validate a novel one-step labeled integrin-targeted tracer,18F-Al F-NOTA-PRGD2 PET/CT for detecting tumor angiogenesis and monitoring the early therapeutic efficacy of antiangiogenic agent Endostar in human nasopharyngeal carcinoma(NPC)xenograft model.Materials and Methods: 18F-Al F-NOTA-PRGD2 was one-step preparation synthesized by chelating reaction using NOTA-PRGD2 and 18F-Al F under 100 ℃.Mice bearing NPC received 18F-Al F-NOTA-PRGD2 by tail vein injection and then in vivo radioactive distribution and small animal PET/CT were studied.For monitoring early response of Endostar antiangiogenic therapy,mice bearing NPC underwent 18F-Al F-NOTA-PRGD2 PET/CT at baseline and after 2,4,7,and 14 days of consecutive treatment with Endostar or PBS,which was compared with 18F-FDG PET/CT as control.Tumors were harvested at all imaging time points for histopathological analysis with H&E and microvessel density(MVD)and integrin avβ3 immunostaining.The correlation between the maximum percent injected dose per gram of body weight(%ID/gmax)tumor uptake of 18F-Al F-NOTA-PRGD2 PET/CT and the corresponding MVD was studied.Results: 18F-AlF-NOTA-PRGD2 was synthesized successfully in short reaction time,with high purity.In vivo body radioactive distribution demonstrates that 18F-Al F-NOTA-PRGD2 can target tumor lesions,and the mean percent injected dose per gram of body weight(%ID/g)tumor uptake at 1h and 2h after the injection of 18F-Al F-NOTA-PRGD2 were 2.14±0.54 and 1.52±0.37,and the tumor/muscle ratio is 3.56±0.36 and 3.12±0.24,respectively.Small animal PET/CT examination also demonstrated the good targeted of 18F-Al F-NOTA-PRGD2 for tumor angiogenesis in NPC model.In terms of monitoring early response of Endostar antiangiogenic therapy,the maximum percent injected dose per gram of body weight(%ID/gmax)tumor uptake of 18F-Al F-NOTA-PRGD2 PET/CT was significantly lower than that of the control group starting from day 2(p < 0.01),which is much earlier and more accurately than that of 18F-FDG PET/CT.Moreover,a moderate linear correlation was observed between tumor MVD and the corresponding tumor uptake of 18F-Al F-NOTA-PRGD2 PET/CT(r = 0.853,P < 0.01).Conclusions: 18F-Al F-NOTA-PRGD2 PET/CT can be used for in vivo angiogenesis imaging and monitoring early response to Endostar antiangiogenic treatment in NPC xenograft model,favoring its potential clinical translation.