Construction And Evaluation Of RGD-USPIO Nanoparticles For Tumor MR Imaging

Part I: Construction and evaluation of RGD-USPIO nanoparticles for tumor MR imaging.Objective: To develop tumor angiogenesis targeted superparamagnetic MR contrast agent,to investigate the cRGD-USPIO binding ability to cells and its application in tumor MR molecular imaging.Method: Ultra-small superparamagnetic iron oxide (USPIO) nanoparticles were prepared by one step chemical co-precipitation method. Tumor angiogenesis targeted superparamagnetic nanoparticles were prepared by covalently conjugating cyclic RGD(c-RGD) containing peptide sequence RGD to USPIO. We used optical microscopy and transmission electron microscopy to observe the nanoparticles'morphology and detect the size of the core of particles. USPIO and cRGD-USPIO particles size distribution were analyzed by laser scattering analyzer. Iron content in cRGD-USPIO was detected by phenanthroline assay. Human lung adenocarcinoma cell line A549 and human umbilical vein endothelial cells (HUVECs) were cultured. cRGD-USPIO and USPIO were incubated with A549 cells and HUVECs respectively for 24h, after 24h ,Prussian blue staining was performed to detect the specific binding ability of cRGD-USPIO to A549 cells and to HUVECs. In vitro, we established A549 xenograft models in nude mice. The nude mice were scanned first. Then USPIO and cRGD-USPIO were intravenously injected to nude mice. After 24h MRI was applied to detect and gather the signal strength. By comparing with USPIO, cRGD-USPIO enhancement to MR signal in diagnosing tumors was evaluated. Result: We successfully developed cRGD-USPIO nanoparticles. The core diameter varies from 5 to 10 nm. The mean diameter of cRGD-USPIO is 43.97±10.1nm. The quality saturation magnetic intensity is 59.94 emu/g and the iron content is 22.88mg/ml.Cell binding ability experiments suggested that compared with the USPIO group, cRGD-USPIO group significantly increased the positive staining. MRI diagnosis in animals showed that reduce the signal strength of USPIO group was 315.76±69.85 and reduce the signal strength of cRGD-USPIO group was 792.17±116.51 which is 2.5 times more than that of USPIO group, compared with the USPIO group, tumor signal was significantly decreased in the cRGD-USPIO group (P <0.01).Conclusion: cRGD-USPIO nanoparticles were successfully prepared. These nanoparticles maintain stable phyiscal and chemical properties. cRGD-USPIO and bound with A549 cells and HUVECs 100% respectively. The cRGD-USPIO nanoparticles we developed could be a novel specific MR negative contrast agent in early tumors diagnosis.Part II: Acute and subacute toxicity test for tumor angiogenesis targeted iron oxide nanoparticles.Objective: To evaluate the toxicity of cRGD-USPIOMethod: 30 Kunmming mice were randomly divided into 3 groups. The mice in one group were injected with saline through tail vein. The second group was injected with low dose of cRGD-USPIO and the third group with high dose of cRGD-USPIO. After injection, external organs, skin changes, activity, food intake, body weight changes were observed. After 2 weeks, retrobulbar venous plexus blood was taken and RBC,WBC,total hemoglobin(HGB) and platelets(Plt) were counted. Automatic Analyzer 7600-110 biochemical analyzer was used to detect ALT,AST,TBIL,UREA,UA,CREA. Then the mice were sacrificed. The organ index was calculated and HE and Prussian blue staining were performed.Result: In our study, Mice were administered with maximum dose. In acute toxicity test, there was no death. We observed that mice in high-dose group had a temporary short time of less activity, while mice in other groups were normal. After 1 week, external organs, skin fur, activity and food intake were normal in all groups. After 2 weeks, retrobulbar venous plexus blood was taken for regular blood test. We observed that there was no significant difference among the three groups. Results of biochemical indicator suggest that there were no significant difference too; there was no difference of weight changes in all groups. HE staining was visible in mice biopsy samples of each group. No edema, degeneration and necrosis were observed in the liver,spleen,kidney,heart and lung cells of mice in each group. In Prussian blue staining, we can clearly observer blue staining of iron in all tissues except the lung of mice in high-dose group.Conclusion: The LD50 of mice injected intravenously with superparamagnetic iron oxide contrast agent > 570mgFe/Kg, much higher than the amount of clinical practice (0.56-0.84mgFe/Kg). We performed pathological and blood test. No change was significantly observed. Our study suggests that cRGD-USPIO is of low toxicity.