| Background:The incidence of breast cancer presents a rising trend through this years. Although the diagnosis and treatment of the breast tumors has achieved tremendous progress, the mortality is still followed after the lung cancer. The rapid development of molecular imaging make the understanding of tumor into a new era. The prominent trait of molecular imaging is to realize the real-time, non-invasive, dynamic, in vivo imaging on molecular cell level, to study the biological processes by qualitative and quantitative research, which has an important application value in the early diagnosis, tumor staging, curative effect evaluation and drug development. Especially the multimodality(double) molecular imaging studies have obvious advantages, which will become the trend of molecular imaging in future. The core content of the multimodality(double) molecular imaging is to construct the ideal multimodality(double) molecular probes which have biological characteristics in vivo. Study the biological behavior of the tumor which target based on HAb18G/CD147 by multimodality(double) molecular imaging may contribute to tumor early detection, early diagnosis, early warning, assessment of therapeutic effect and prognosis of metastatic evaluation etc..Objective:1. To prepare Dual-modality Single-Photon Emission Computed Tomography(SPECT) and magnetic resonance(MR) molecular nanoprobes 125I-SPIO-HAb18F(ab′)2 targeting HAb18G/CD147, which is expressed on breast cancer cell membranes and to investigate the physicochemical and biological properties in vitro. 2. Culture human cell lines MDA-MB-231 and MDA-MB-468. To investigate the expression and significance of HAb18G/CD147 protein on the cell surface, and to study the dual mode probes’ cytotoxicity in vitro. 3. Preliminary study the targeting and specificity of dual mode probe to the breast cell and to explore its biodistribution in normal mice.Method :1. Superparamagnetic iron oxide nanoparticles(SPIOs) were prepared by “one-pot reaction” method as described. The single-chain antibody fragments HAb18F(ab’)2 were conjugated to SPIOs via chemical method and then labeled with 125 I using Iodogen method. The final 125I-SPIO-HAb F18(ab’)2 nanoprobes were purified. SPIOs and 125I-HAb18F(ab′)2 were used as control in this research. We carried preliminary evaluation on their physicochemical properties and biological characteristics in vitro: Transmission electron microscope(TEM) and dynamic light scattering(DLS) were used to measure these nanoparticle sizes and the hydrodynamic diameters. Applied of paper chromatography to calculate the labeling rate and used the thin layer chromatography to determination the radiochemical purity of 125I-SPIO-HAb18F(ab′)2 and 125I-HAb18F(ab′)2. The parallel stability experiment in vitro and lipid water partition coefficient experiment were also to investigate. The MRI T2 transverse relaxation efficiency(r2) of the SPIO and 125I-SPIO-HAb18F(ab′)2 at different Fe2+concentrations(3.0×10-2, 2.0×10-2, 1.5×10-2, 7.5×10-3, 3.75×10-3 mmol/l) were measured with 1.5 Tesla clinical MR scanner. 2. The human cell lines MDA-MB-231(experience group) and MDA-MB-468(control) were cultured. The expression of HAb18G/CD147 in breast cancer cell lines were determined by flow cytometry. The target probe 125I-SPIO-HAb18F(ab′)2 or non-target probe SPIO with MDA-MB-231 cells were put togther to incubated. The morphology and distribution of the targeted group probe inside and outside of the cells were observed, and to compare with the non-targeted group by transmission electron microscopy. The effects of 125I-SPIO-HAb18F(ab′)2 and SPIO on the proliferation of MDA-MB-231 and MDA-MB-468 cells were investigated by MTT method. 3.(1) 125I-SPIO-HAb18F(ab′)2 with MDA-MB-231 or MDA-MB-468 cells and SPIO with MDA-MB-231 or MDA-MB-468 cells were cultured. The untreated MDA-MB-231 cell suspension were used as control group. All of the above samples were detected by MRI. Then compared the MR signal intensity between the differences groups, and calculated the average change rate of the MRI signal intensity.(2) The specificity binding experiment: Used the same method as mentioned above to culture 125I-SPIO-HAb18F(ab′)2 with MDA-MB-231, MDA-MB-468 cell and cultivate SPIO with MDA-MB-231, MDA-MB-468, respectively. After centrifugation, supernatant and repeated PBS flushing at different times(20min、40min、1h、2h、4h), then measured the radioactivity of each tube using γ-counter. The cell binding rate were calculated and did statistical analysis.(3) Took 125I-HAb18F(ab′)2 as the radioligand, then carried on cell membrane associated antigen competitive inhibition experiment to determination the half maximal inhibitory concentration of SPIO-HAb18F(ab′)2 and HAb18F(ab′)2.(4) 32 normal mice were randomly divided into 2 groups, they were injected 125I-SPIO-HAb18F(ab′)2 and 125I-HAb18F(ab′)2 through tail vein respectively. Dissected the organs and blood of mice at different time(15min、6h、24h、48h) to measure the biodistribution of radioactivity and did plasma clearance test to calculate the half-life of plasma.Results:1. The dual-modality probe targeting HAb18G/CD147 was successfully constructed. The SPIOs were fairly homogeneous with an average core size of(10.32 ± 1.3) nm; the SPIO and 125I-SPIO-HAb18F(ab’)2 hydrodynamic diameter of 44.80 nm and 52.64 nm. The radio chemical yield(RCY) of 125I-SPIO-HAb F18(ab’)2 and 125I-HAb18F(ab’)2 were 41.9% and 85.8%, respectively; the radiochemical purity(RCP) of the two groups were more than 95%, suggesting well stability in vitro; The lipo-hydro partition coefficient values were(-0.99 ± 0.03) and(-1.49 ± 0.08), respectively, which demonstrated that they were all water-soluble substances. MRI scanning showed that the transverse relaxation efficiency(r2) of SPIO and 125I-SPIO-HAb18F(ab’)2 were 38.79 m M-1·s-1 and 106.73 m M-1·s-1, respectively. 2. The human breast cell lines MDA-MB-231 and MDA-MB-468 were growing at monolayer. The results of the flow cytometry confirmed that HAb18G/CD147 had high level expression on the plasma membrane of MDA-MB-468, while the MDA-MB-468 had low level. The mean fluorescence intensity were 1353 and 162, the former is about 8.35 times to the latter. The results of the transmission electron microscope showed the treatment group nanoparticles gather together near the cell membrane and cytoplasm, which the diameter was about 20 nm. The control group nanoparticles were around the outside of the cells, no regularity and was far away from the cells. MTT assay showed that 125I-SPIO-HAb18F(ab′)2 and SPIO had no obvious effect on both MDA-MB-231 and MDA-MB-468 cells when iron concentration was ≤40μg/ml. 3.(1) In vitro MRI results showed: the MDA-MB-231 cells with 125I-SPIOHAb18F(ab′)2 incubated together presented apparent shorter T2 signal, other control groups and the blank group showed obviously high signal, there had statistical differences among them(P<0.05).(2) The specificity binding experiment showed the binding rates between experimental and control group had statistically significant(P<0.05).(3) The competitive inhibition experiment showedSPIO-HAb18F(ab′)2 had high affinity with the membrane associated antigen HAb18/CD147, its IC50 value was 0.48 nmol/L, while the IC50 value of HAb18F(ab′)2 was 1.60 nmol/L.(4) The biodistribution in normal mice showed: the half-life of plasma of 125I-SPIO-HAb18F(ab′)2 was 8.85 h, which was mainly concentrated in the liver, spleen and excretion by the kidneys. In 10 min, the liver %ID/g value was(19.10 ± 0.76), the spleen was(14.04 ± 1.49), the kidneys was(2.62 ± 0.61). With the extension of time, the %ID/g value was reduce. 125I-HAb18F(ab′)2 was aggregated in blood and the half-life of plasma was 10.38 h.Conclusion:1. The dual-modality probe targeting HAb18G/CD147 was successfully constructed with good physicochemical properties and in vitro biological characteristics, which has the potential for clinical application. 2. The dual-modality probe had high label rate on positive expression about membrane associated antigen and had strong targeting, may be a potential molecular imaging agent for MR imaging and SPECT for the early detection of Breast Cancer. |
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