Preparation And Characterization Of Dual-modality Molecular Probes For MRI And Near-infrared Fluorescence Imaging Of Gastric Cancer Vasculature

Background:The incidence of gastric cancer stands the second position in malignant tumor in theworld,and the diagnosis and treatment of tumor is still faces enormous challenges. Atpresent, the imageological examination applied to clinical is difficult to realize the earlydiagnosis, monitoring of metastasis and prediction of treatment effect. In recent years, therapid development of molecular imaging and nanometer particle preparation technologynot only contribute to the early diagnosis of tumor, and can realize the visualization andquantitation of tumor biological behavior in cellular and molecular level. Angiogenesisplays a pivotal role in tumor initiation, growth, and metastasis. Identification of a ligand/receptor system that enables functionalized nanoparticles to efficiently target cancer holdsgreat promise for the development of novel approaches for the detection and treatment ofmalignant tumor.Objective:1) To develop and characterize an MR/optical dual-modality probe targetingangiogenesis for imaging and treating gastric cancers.2) We established BGC-823xenograft models in nude mice,then intravenously injected DPs and Fe3O4-Cy5.5to explore its applicable value as a contrast agent fordual-modality imaging.Methods:1) We conjugated GX1peptide, a novel gastric cancer neovasculature targeted peptideidentified by phage-display technology, with near-infrared fluorescence dye, Cy5.5,resulting Cy5.5labeled GX1(Cy5.5-GX1). Cy5.5-GX1was directly conjugated to thesurface functionalized magnetic Fe3O4nanoparticles. The hydrodynamic size and zetapotential of Fe3O4@PEG-COOH, Fe3O4-Cy5.5and DPs were analyzed by nano-ZS.2) To investigate the effects of DPs on the proliferation of HUVECs and BGC-823cellsby MTT method. DPs with different concentration was scanned on different MRsequences, T1，T2and T2*WI.3) We established BGC-823xenograft models in nude mice,then intravenously injectedDPs and Fe3O4-Cy5.5. MRI was applied at different time points to follow the changesof signals. For the evaluation of the MR images, the tumor area of the two groups wasdivided into three ROIs and the percentage of reduced pixels (pre vs post) was plottedagainst the corresponding ROI. At the same time, we observe the time-resolvedaccumulation of the nanoparticles in the tumors by using in vivo fluorescence imagingwithin a period of48hours. Then tumor and normal tissues were collected, and exvivo optical imaging was performed on the tissues. The frozen tissue sections wereexamined by Prussian blue staining and immunohistochemical staining.Results：1) DPs have excellent stability in PBS; The hydrodynamic size of Fe3O4@PEG-COOHis35.23±0.07nm and the zeta potential is0.31±0.20mV. The hydrodynamic size ofFe3O4-Cy5.5is38.23±0.06nm and the zeta potential is0.29±0.60mV, and thehydrodynamic size of DPs is39.49±0.16nm and the zeta potential is-4.15±0.79mV.The coupled rate of DPs with polypeptide is＞90%.2) MTT assay showed that DPs had no obvious effect on both HUVECs and BGC-823cells when iron concentration was≤150μg/ml.3) The signal intensity increased first and then decreased following the increasing concentrations of DPs on T1weighted image (T1WI)，especially on FSPGR-T1WI(P<0.01). There were no significant differences in signal intensity on FSE-T2WI andSSFSE-T2*WI when iron concentration＞10μg/ml and signal intensity onFESITA-T2*WI was significantly lower than the former grou（pP＜0.05），while wheniron concentration was≤10μg/ml， the signal intensity on SSFSE-T2*WI wassignificantly decreased compared to FESITA-T2*WI (P<0.05)．4) MRI showed that after injecting DPs in the experimental group, the contrast to noiseratio(CNR) of xenograft at8h and12h decreased comparing with the value beforeinjection (F=49.55, P＜0.01); In contrast, there was no apparent signals change in thetumors of mice receiving Fe3O4-Cy5.5(F=2.306, P＞0.05). The decrease of MRsignals was prevailingly confined to the periphery of the tumors receiving DPs andFe3O4-Cy5.5(t=-7.872,t=6.678;P＜0.01). The tumor of experimental group showedthe high contrast to background tissue during2.0–48h p.i, but there was no significantdifference in fluorescence intensity between tumor and background tissue of micereceiving Fe3O4-Cy5.5. Furthermore, ex vivo evaluation of excised organs showedDPs was predominantly taken up by the tumor and lung at48h p.i. Iron-positive cellswere detected in the liver and spleen of the two groups and in the tumor of the micethat received DPs. The normal kidney tissues of the two groups and the tumor in themice receiving Fe3O4-Cy5.5were consistently negative for iron staining.Conclusions：1) The dual-modality probe targeting angiogenesis of gastric cancer wassuccessfully constructed.2) DPs can selective deliver into Gastric Cancer and this novel receptor-targetednanoparticle may be a potential molecular imaging agent for MRI and NIRFoptical imaging for the early detection of Gastric Cancer.