Establishment Of A Gastric Cancer Specific Magnetic/optical Dual-modality Probe And Imaging Study

【Background】Gastric cancer is one of the malignancies that severely threatens human health.Delayed diagnosis attributed to its high mortality rate, thus early detection is the key to improve survival. However, current clinical methods are ineffective in early diagnosisbecause of lacking sensitivity and specificity. MGb2is a gastric cancer specificmonoclonal antibody developed by academician Daiming Fan in our laboratory. It hasgreat potential to be a gastric cancer diagnostic marker with high binding specificity andaffinity to gastric cancer cells. Molecular imaging, being a new tool in basic research,could detect molecule changes in a continuous and kinetic fashion and has promisingfurther in clinical use. Multi-modality imaging could complement each modality andacquire more precise and detailed information, providing more solid evidence in diseasediagnosis.【Aim】To establish a gastric cancer specific, magnetic/optical dual-modality probe usingmonoclonal antibody MGb2as guiding molecule; to characterize the probe and verify itsgastric cancer imaging ability in in vitro and in vivo settings.【Method】1. Synthesize and characterization of the dual-modality probe:Fe（acac）3, PEG（COOH）2and oleylamine were used to synthesize biocompatibleFe₃O₄magnetic nanoparticle （MNP） via hyperthermal decomposition method. Twoconjugating strategies were proposed for probe establishment as1) parallel: conjugatingMGb2antibody and Cy5.5NH2dye simultaneously onto MNP surface and2) paiallel:label MGb2with Cy5.5NHS dye and then conjugate the antibody onto MNP surface.Transmission electron microscopy （TEM） was used to determine MNP size, dynamic lightscattering （DLS） to test MNP hydrosize and Zeta potential, thermal gravimetric analysis（TGA） to analyze MNP organic content, magnetometer to test saturation magnetizationand UV-Vis absorbance and fluorescence excitation assays to check the results of antibodylabeling. Reaction pH and feed ration of materials were modified according to DLS andfluorescence excitation results. Relaxation rate （r₂） of the probe was determined bymagnetic resonance scanning and T2mapping calculation. Bicinchoninic acid （BCA） assaywas used to quantify antibody and XTT assay to identify probe’s toxicity.2. In vitro imaging study: In vitro studies, UV-Vis, fluorescence excitation and laser confocal microscopyassays were used to test probe’s optical character and tumor specificity; Prussian’s bluestain and magnetic resonance scanning with T2mapping calculation to test probe’smagnetic character.3. In vivo imaging study:In vivo studies, nude mice with gastric cancer xenograft were used as animal model;different probes （specific probe, unspecific probe and MGb2+specific probe） wereinjected through tail vein; optical and magnetic images were acquired at different timepoints after probe injection by IVIS optical imaging system and Bruker animal magneticresonance imaging instrument.【Results】1. Characterization of the probes:Fe₃O₄MNP was synthesized by hyperthermal decomposition method. Its TEMdiameter is16.7±1.9nm, hydrosize is42.6±0.5nm, Zeta potential is6.4±0.3mV,organic content is68.9%and saturation magnetization is55.7emu/g. UV-Vis assay showsone MGb2antibody is labeled with3.3Cy5.5dye molecule. Parallel probe has ahydrosize of77.3±1.1nm and Zeta potential of-19.6±1.2mV; while paiallel probe’shydrosize is87.7±0.3nm and Zeta potential is-19.3±0.5mV. Through parallel strategy,one MNP could be conjugated with1.4MGb2antibodies while paiallel strategy couldincrease this number to3. Fluorescence excitation assay reveals that at4*10-2mg/ml Feconcentration, parallel probe’s fluorescent intensity is5and paiallel probe’s is10.Magnetic resonance scanning and T2calculation suggests the probe’s relaxation rate is231.1mM-1s-1. XTT assay demonstrates that the probe exhibits very low cell toxicitybelow0.3mg/ml Fe concentration. At the concentration of0.3mg/ml Fe, viable count ofcells is85.3%of negative control.2. In vitro imaging study:SGC-7901and GES cells were subjected to fluorescence excitation assay afterincubation with probes, results shows that, in parallel probe group, SGC-7901cells havehigher fluorescence output than GES cells （0.117±0.019vs.0.029±0.026）; similar results are obtained in paiallel probe group. Besides, incubated at the same concentration,paiallel probe emits more fluorescence than parallel probe （0.472±0.138vs.0.117±0.019）, suggesting paiallel probe has better optical imaging ability. Thus paiallel probewas used in the following experiments. In vitro near-infrared imaging could alsodistinguish SGC-7901cells with GES cells by different image signal. Laser confocalmicroscopy assay shows the probe could specifically bind to SGC-7901cells’ surface butnot GES cell’s surface. Prussian’s blue stain and magnetic resonance imaging could alsowell discriminate the two cells that SGC-7901cells have a much lower T2value comparedwith GES cells （60.5±13.4vs.526.8±120.3）.3. In vivo imaging study:In vivo studies, various probes were injected into nude mice through tail vein with a15mg Fe/kg dosage. Living animal optical imaging displays that the tumor is best imagedat24h after specific probe injection （9.30*10⁸±2.04*10⁸p/s/cm²/sr）. Then the signalattenuates and become a little higher than pre-injection condition at the72h time point（3.16*10⁸±5.40*107p/s/cm²/sr）. The unspecific probe group’s signal is slightly increasedand quickly restored to normal. Same tendency is detected by magnetic resonance imagingand T2*calculation. At the time point of6h,24h,48h and72h after probe injection, theT2*value of tumor region is86.91%,68.95%,82.37%and90.17%of the same regionbefore probe injection. In the negative control group, these data are97.39%,90.17%,95.88%and101.59%and in the competition group, the T2*values of tumor region are91.83%,85.90%,98.77%and106.57%of pre-injection conditions. Biodistribution assayimplicates that the probe is metabolized mainly through liver.【Conclusion】Gastric cancer specific, optical/magnetic dual-modality probe Fe₃O₄-MGb2-Cy5.5issuccessfully established. The probe has satisfactory imaging capability andbiocompatibility that could bind and detect gastric cancer cells specifically and sensitively.It is a promising probe for clinical gastric cancer early diagnosis and has great potential tobe clinical available. Further modification, such as coupling the probe with anti-cancerdrugs, could expand its application and achieve the theranostic goal.