Molecular Target Nano Iron Oxide Particles Of Magnetic Resonance Imaging Of Pancreatic Cancer Cells

OBJECTIVE:To establish a magnetic resonance molecular conjugator of chemokine receptor4(CXCR4) monoclonal antibody and ultrasmall superparamagnetic iron oxide particles (USPIO); and to evaluate the expression levels of CXCR4in human pancreatic cancer cells including AsPC-1, BxPC-3, CFPAC-1, PANC-1, pancreatic adenocarcinoma tissues and normal pancreatic tissues; and to investigate targeted combination specificity and MRI targeted imaging feasibility of CXCR4-USPI0to the four human pancreatic cancer cells; and to study the relationship between in vitro MR T2value enhancement ratio and cellular CXCR4expression level.Methods:CXCR4monoclonal antibody and bull serum albumin (BSA) were respectively bioconjugated with USPIO using carbodiimide [1-ethyl-(3-dimethylamino propyl) carbodiimide hydrochloride (EDC)]. MTS cytotoxicity test was carried out to assess the safty of USPIO conjugators. Transverse relaxation time (T2value) and free induction decay time (T2*value) differences of CXCR4-USPI0, BSA-USPIO conjugator and USPIO solutions were evaluated at the same iron concentration grades. Western blot test was used to semi-quantitatively analyze the expression levels of CXCR4in four pancreatic cancer cells. Immunofluorescence and immunohistochemisty techniques were performed to detect the expression sites and levels of CXCR4in four pancreatic cancer cells, pancreatic adenocarcinoma tissues and normal pancreatic tissues. After incubating four pancreatic cancer cells with CXCR4-USPI0and BSA-USPIO respectively, changes of T2and T2*values were measured using a clinical1.5T MRI scanner, and distribution of iron particles in the incubation solution was observed by Prussian blue staining.Results:CXCR4-USPI0and BSA-USPIO nanoparticles were successfully established, and MTS result revealed that Cell Relative Viability was over80%at a range of6.25to200μgFe/ml of iron particle concentration. MR scan proved that there was no significant T2or T2*value difference between CXCR4-USPI0and USPIO groups at every specific iron concentrations. And there was linear correlation between△R2/△R2*values and iron particle concentrations. According to the result of Western blot test, CXCR4peptide located at the marker band with molecular weight of34KDa. The peptide grey value ratio of CXCR4band versus p-actin band of AsPC-1, BxPC-3, CFPAC-1, PANC-1cells were0.62,0.95,0.89,0.72respectively. Immunofluorescence result revealed that CXCR4expression was positive both on the membrane and in the cytoplasm of all four pancreatic cancer cells. While mean fluorescence signal intensity of AsPC-1, BxPC-3, CFPAC-1, PANC-1cells were23.66±17.04,61.00±25.04,58.74±18.40,27.48±21.44respectively. Immunohistochemisty result showed CXCR4was highly expressed in pancreatic adenocarcinoma tissues, whereas no obvious CXCR4expression had been detected in normal pancreatic tissues. After incubation with four pancreatic cells, T2and T2*values of CXCR4-USPI0group was lower than BSA-USPIO group. In CXCR4-USPI0group, T2value enhancement ratio of AsPC-1, BxPC-3, CFPAC-1, PANC-1were54.92%,85.36%,80.16%and56.95%respectively, which has a good correlation with the peptide grey level of CXCR4band measured by Western blot (r=0.976, p=0.024) and fluorescence signal intensity detected by Laser scanning confocal microscope (r=0.996, p=0.004). More iron particles were found in CXCR4-USPI0group as shown in Prussian blue staining. Distribution of iron particles was mainly around the cell membrane in CXCR4-USPI0group, while scattered iron particles were found in the inter-cell space in BSA-USPIO group.Conclusion:CXCR4-USPI0conjugator had satisfied targeted combination specificity and MRI targeted imaging feasibility according to CXCR4positive pancreatic cancer cells. T2value enhancement ratio could partially reveal the expression level of CXCR4protein.