A Novel Radiolabelled CRGD-conjugated Ultrasmall Superparamagnetic Iron Oxide Nanoparticles For Dual-modality Imaging And Treatment For Breast Cancer

Part I The preliminary experiment of the effect of radioiodinated novel cyclicRGD on the diagnosis of breast cancer Objective: To evaluate the targeted diagnostic capability of radioiodinated novel cyclicRGD by testing its cell uptake, biodistribution in tumor bearing nude mice and SPECT imaging. Methods: A novel cyclicRGD dimer was designed by ourselves and radiolabelled with 125 I using C hloramine-T method. The expression of integrin receptor ανβ3 on surface of human breast cancer cell line Bcap37 was determined by immunohistochemical staining. The specific binding of ¹²⁵I-cRGD on breast cancer cell was detected by cell uptake. 24 female nude mice were given an i.v. injection of radio iodinated cRGD at a dose of approx. 370 k Bq via the tail vein. Tissues, including blood, liver, kidneys, tumor, small intestine, etc., were obtained at intervals, weighed, and counted for radioactivity. The percentage of injected dose per gram of wet tissue（% ID/g tissue） was calculated. Bcap37-bearing mice were injected with ¹²⁵I-cRGD each via the tail vein. The SPEC T scans were carried out at various time points thereafter. The energy peak for the camera was set to 37 ke V, and the energy window was set to peak energy ± 30%. The scintigraphic results were evaluated visually and analyzed quantitatively. Results: RGD peptides, CC-16: c（Cys-Arg-Gly-Asp-d Ser-Cys）-Tyr-d Ser-Lys-Tyr-c（Cys-ArgGly-Asp-d Ser-Cys）（MW:1798.98）, were synthesized. The result of paper chromatography showed that（99.24±0.47）% of 125 I was bound to cRGD. The radiochemical purity of the labeled compound remained 97% at 48 h in human blood serum and physiological saline.Integrin receptor ανβ3 was positively expressed on breast cancer cell line Bcap37. The uptake of ¹²⁵I-cRGD on Bcap37 were（2.89±0.52）%,（3.11±0.15）%,（3.56±0.39）%,（4.35±0.56）%, and（4.50±0.68）%,at incubation time of 1, 2, 4, 8 and 24 h, respectively. ¹²⁵I-cRGD uptake increased with time（rs=0.858,P<0.01）. Accumulation of radioactivity in tumor reached a maximum of approximately 2.33% ID/g at 4 hour after injection and decreased slowly with time. At 4 hour after injection, radioactivity in tumor was higher than other organs except kidney. Tumor/muscle ratios increased over time and the ratio reached 6.13±2.77 at 24 h after injection. Tumors were clearly visualized almost from the start of SPECT imaging after injection. There was no accumulation of ¹²⁵I-cRGD in the opposite forelimb. In later static images, the background radioactivity decreased, while radioactivity in tumor increased. Conclusions: Radioiodinated novel cyclicRGD could specially bind to integrin receptor ανβ3 on surface of human breast cancer cell line Bcap37, and could be used as a promising radiotracer for detection of breast cancer.Part II Radioiodinated cRGD-USPIO as MRI agent forimaging of breast cancer Objective: To evaluate the targeted diagnostic capability of radioiodinated novel cyclicRGD conjugated USPIO as MRI agent. Methods:cRGD peptide was conjugated to the carboxyl group on the surface of CMD-USPIO through the actions of dehydration reagents DCC and NHS. cRGD-USPIO was radiolabelled with 125 I using C hloramine-T method. Transmission Electron Microscope（TEM） imaging of CMD-USPIO and 125-cRGD-USPIO were obtained. The average hydrodynamic diameter of the samples was determined by dynamic light scattering（DLS）. Cells were incubated with nanoparticle solution for 4 h and then harvested. Subsequently, the cells were transferred to agar gelatin in 2-m L Eppendorf tubes and measurements were taken at room temperature. The relaxivity of ¹²⁵I-cRGD-USPIO and CMD-USPIO was measured. The uptake of ¹²⁵I-cRGD-USPIO and CMD-USPIO was also assessed histologically using Prussian blue staining. An MRI scan was performedusing a 3.0-T whole-body clinical scanner at various time points after injection. Tumor tissues were also studied by TEM images. Results: The average hydrodynamic size of CMD-USPIO dispersed in cell culture media was 32.61 nm, and the hydrodynamic size of ¹²⁵I-RGD-USPIO was 53.67 nm. The result of paper chromatography showed that（99.49±0.24）% of 125 I was bound to cRGD-USPIO. The results of Prussian blue staining demonstrated a strong uptake of ¹²⁵I-cRGD-USPIO but a weak uptake of CMD-USPIO in the Bcap37 cells post incubation. Blocking the ανβ3 integrin receptor with free RGD effectively reduced the amount of blue staining in the Bcap37 cells. MRI signal intensity of Bcap37 cells in gelatin incubated with ¹²⁵I-cRGD-USPIO decreased significantly compared with cells incubated with CMD-USPIO. The tumor presented high signal intensity on MRI T2 WI before the administration of radiolabeled conjugates. A negative contrast enhancement was observed in the tumor after intravenous administration. Four hours after the administration of ¹²⁵I-cRGD-USPIO, the Bcap37 tumor exhibited a low- intensity signal in various areas of the tumor on T2 WI. TEM images showed a certain amount of ¹²⁵I-RGD-USPIO probe accumulated in lysosome. Conclusions:¹²⁵I-cRGD-USPIO showed high uptake in breast cancer and could be used as a promising agent for detection of breast cancer by MRI. Part III Radioiodinated cRGD-USPIO as a radiotracer for imaging of breast cancer by SPECT Objective: To study the application of ¹²⁵I-cRGD-USPIO in SPECT for targeted diagnosis of breast cancer using a small-animal model. Methods: CMD-USPIO was radiolabelled with 125 I using Chloramine-T method. To compare cell uptake of ¹²⁵I-CMD-USPIO and ¹²⁵I-cRGD-USPIO in human breast cancer cell line Bcap37. The SPEC T scans were carried out at various time points using ¹²⁵I-CMD-USPIO or ¹²⁵I-cRGD-USPIO. Female Bcap37-bearing nude mice were given an i.v. injection of¹²⁵I-CMD-USPIO or ¹²⁵I-cRGD-USPIO via the tail vein. Tissues, including blood, liver, kidneys, tumor, small intestine, etc., were obtained at intervals, weighed, and counted for radioactivity. The percentage of injected dose per gra m of wet tissue（% ID/g tissue） was calculated. Results: The labelling efficiency of ¹²⁵I-CMD-USPIO was（30.79±15.02）%. The result of paper chromatography showed that（98.25±0.71）% of 125 I was bound to CMD-USPIO. High radiochemical stability of ¹²⁵I-cRGD-USPIO was found up to 96 h in fresh human serum and physiological saline. However, the stability in physiological saline was slightly lower than in human serum at 72 and 96 h. The uptake of ¹²⁵I-cRGD-USPIO increased in the first 4 h and then decreased. The uptake of ¹²⁵I-CMD-USPIO was lower than that of ¹²⁵I-cRGD-USPIO at various time point（P<0.01）. The radioactivity of ¹²⁵I-CMD-USPIO mainly accumulated in liver and spleen on SPECT images. Animals were subjected to SPECT imaging with ¹²⁵I-cRGD-USPIO. Tumors were clearly visualized almost from the start of SPECT imaging after injection. There was no accumulation of ¹²⁵I-cRGD-USPIO in the opposite forelimb. In later static images, the background radioactivity decreased, while radioactivity in tumor increased. The use of ¹²⁵I-cRGD-USPIO provided good contrast in the images. The biodistribution study showed that the radioactivity levels in tumor increased during the first 4 h following injection, whereas those in tissues, including liver, spleen, lung, kidney, and stomach, decreased with blood levels. Accumulation of radioactivity in tumor reached a maximum of（8.08 ± 0.3） %ID/g after 4 h. Tumor/muscle ratios increased over time and the ratio reached 9.45 ± 4.03 at 96 h after injection. Conclusions: ¹²⁵I-cRGD-USPIO showed a long circulation half- life, high tumor uptake, and high initial blood retention with moderate liver uptake, making it an attractive SPECT/MRI dual-modality agent for the detection of breast cancer.Part IV An experimental study on targeted therapy of radioiodinated cRGD-USPIO on breast cancer Objective: To study the inhibitory effect of ¹³¹ I-cRGD-USPIO in Bcap37-bearing mice and prove its inhibitory effect to tumor angiogenesis by testing the expression of CD31 determined by immunohistochemical staining. Methods: cRGD-USPIO was radiolabelled with 131 I using C hloramine-T method under the optimum labeling conditions. The inhibitory effects of different treatments on the proliferation of human breast carcinoma cell line Bcap37 were measured by MTT assay. The apoptosis rate was assayed by flow cytometry.The human breast carcinoma Bcap37 cells were implanted into nude mice and tumor-bearing nude mice were randomly divided into 4 groups.Drugs were injected into the tail vein of the nude mice.The size of tumors were measured every 2 days and the mice were killed at 28 week after the first injection.The inhibitory rate of tumor growth was calculated and tumor apoptosis was observed by hematoxylin-eosin（HE） staining. The K i67 and CD31 expressions in the xenograft tumors were analyzed by immunohistochemical staining. Results: The tumor inhibitory rate of Bcap37 cells was significantly higher in ¹³¹ I-cRGD-USPIO group than those in other groups. The apoptosis rate in the study group was higher than those in other positive control groups and the blank control group, respectively. When sacrificed at 28 th day, the tumor sizes were（9803.95±665.52） mm3 for control group,（9935.05±1200.37） mm3 for 131 I group,（6827.49±606.83） mm3 for cRGD-USPIO group, and（5597.65±724.15） mm3 for ¹³¹ I-cRGD-USPIO group. Significant differences were observed between ¹³¹ I-cRGD-USPIO group with other groups（P<0.01）. Compared with the control group at 28 th day, the tumor inhibitory rates were（26.12±8.37） % for cRGD-USPIO group and（43.07±3.74） % for ¹³¹ I-cRGD-USPIO group respectively. The expressions of K i67 and CD31 were markedly decreased in ¹³¹ I-cRGD-USPIO group compared with control group.Conclusions: ¹³¹ I, cRGD and cRGD-USPIO all could inhibit the growth of human breast carcinoma cell Bcap37 individually. ¹³¹ I-cRGD-USPIO showed more obvious antitumor effect on breast cancer than other drugs. The action mechanism maybe associated with anti-angiogenesis in tumor.