Construction and in vitro characterization of dual-modality single-photon emission computed tomography - MRI nanoprobes targeting HAb18G/CD147 in breast tumors

Abstract

Objective: To prepare dual-modality single-photon emission computed tomography (SPECT)-MRI molecular nanoprobes targeting HAb18G/CD147 expressed on breast cancer cell membranes and investigate the physicochemical and biological properties in vitro. Methods: Superparamagnetic iron oxide nanoparticles (SPIOs) were prepared by one-pot reaction method as described. The single-chain antibody fragments HAbF18(ab') 2 were conjugated to SPIOs via chemical method and then labeled with 125I using Iodogen method. The final 125I-SPIO-HAbF18(ab') 2 nanoprobes were purified. SPIOs or 125I-HAbF18(ab') 2 were used as control. 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. The MRI T 2 transverse relaxation efficiency of these nanoprobes at different Fe2+ concentrations were measured with 1.5 T clinical MR scanner. The 125I-SPIO-HAbF18(ab') 2 and 125I-HAbF18(ab') 2 radiochemical purity were measured by thin layer chromatography and the radio chemical yield was calculated. We also conducted stability tests in vitro and octanol/water partition coefficient experiments. Two breast tumor cell lines, MDA-MB-231 (HAb18G-overexpressing cells,experimental group) and MDA-MB-468 (control), were used for assessment of cells viability at different Fe2+ concentrations (1, 5, 10, 20, 40 μg/ml) by methyl thiazolyl tetrazolium assay. Specific binding experiments in vitro included two parts:magnetic resonance imaging and radionuclide tests, the above-mentioned breast cancer cell lines were incubated with 125I-SPIO-HAbF18(ab') 2 nanoprobes respectively and took MDA-MB-231 cells which were not treated as blank group. First comparing the MR signal intensity differences among experimental group, the control group and blank group, then calculated the rate of MRI signal changes; Two breast tumor cell lines, MDA-MB-231 and MDA-MB-468 were incubated with 125I-SPIO-HAbF18(ab') 2 nanoprobes too, then measured radioactivity counting by γ counter at different time and calculated the cell binding rates, and did statistical analysis by using one-way ANOVA. Results: The SPIOs were fairly homogeneous with an average core size of (10.32±1.30) nm;the SPIO and 125I-SPIO-HAbF18(ab') 2 hydrodynamic diameter of 44.80 and 52.64 nm, and MRI scanning showed that the transverse relaxation efficiency of SPIO and 125I-SPIO-HAbF18(ab') 2 were 38.79 and 106.73 mM-1 · s-1, respectively. The radio chemical yield of 125I-SPIO-HAbF18(ab') 2 and 125I-HAbF18(ab') 2 were 41.90% and 85.50%, respectively. The radio chemical yield of the two groups were >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 both water-soluble substances. Different Fe2+ concentrations (1,5,10,20,40μg/ml) of 125I-SPIO-HAbF18(ab') 2 on breast cancer cell lines MDA-MB-231 and MDA-MB-468 showed no significant inhibition of cell proliferation (F values were 0.78, 0.66; P values were 0.58, 0.66). The cell-specific binding experiment showed: MRI signal intensity values on experimental group, the control group and the blank group were (1 670 ± 5), (1 930 ± 8), (2 349 ± 14), respectively, significant differences existed among these groups (F=4 408.48, P=0.000), the rate of signal intensity change of experimental group and the control group were 28.87%, 17.78%. SPECT:MDA-MB-231 could uptake 125I-SPIO-HAbF18(ab') 2 , the cell binding rates were (6.52 ± 0.60)% and (10.52 ± 2.04)% in 20 min and 4 h, respectively. Conclusions: Our results suggested that the dual-modality SPECT-MRI nanoprobes 125I-SPIO-HAbF18(ab') 2 were prepared successfully with good physicochemical properties and biological characteristics in vitro. These dual-modality molecular imaging nano-probes may have potential to improvearly detection and diagnosis of HAb18G/CD147-expressing cancers and to facilitate the development of HAb18G/CD147-directed interventions.