Construction And Performance Of Multifunctional Imaging Molecular Probe For Pancreatic Cancer:a Preliminary Study

Objective:To constract a kind of molecular imaging probe with multifunctions that of magnetic resonance imaging and pancreatic cancer gene silencing with chemical synthetic method.Further,to evaluate the biological characteristics,imaging conditions and therapeutic effects on pancreatic cancer of this molecular probe in vivo and in vitro.Methods:1.Construction of PEI/SPION gene vector.The organic phase SPIO nanocrystals with uniform and controllable size were prepared by organic high temperature decomposition method,the particle size and distribution were characterized through dynamic light scattering（DLS）,the distribution and interplanar spacing of nanocrystals were observed through transmission electron microscope（TEM）.Branched PEI（MW:2 k Da）was grafted by cholesterol with the assistant of carbonyldiimidazole（CDI）,pure products（PEI-g-Chol）were obtained as gummy solids by lyophilizating.Then,the chemical structures of these products were confirmed by ¹H NMR.The cytotoxicity was detected by CCK-8assay.The SPIO nanoparticles were redispersed in chloroform and mixed with PEI-g-Chol,PEI-g-Chol/SPIO nanoclusters could be obtained by dispersing the above mixture into water.Size distributions,zetapotentials,packing efficiencies and stabilities on siRNA of those nanoclusters were characterized.T₂relaxation times were also measured by a 1.5T magnetic resonance system.2.Characterizations of PEI/SPION/siRNA multifunctional imaging molecular probe in vitro.SiRNA sequence with the best silencing effect was screened by human Mucin 4（MUC4）m RNA sequence.PEI/SPION and siRNA binding efficiency was studied by agarose gel electrophoresis analysis.SiRNA release effect was studied by heparin sodium release assay.PEI/SPION/siRNA molecular probe was also characterized through serum stability assay and phagocytosis assay.Liposomes packaging PEI/SPION was used constracting group to transfect pancreatic cancer cell lines.And then,the expression of MUC4 was detected by RT-PCR and Western blotting methods.3.To evaluate PEI/SPION/siRNA multifunctional imaging molecular probe in vivo,the pancreatic cancer model xenograft tumors on nude mice were established.Tumor growth conditions were observed after the in situ injection of PEI/SPION/siRNA.The expression of MUC4 protein in tumor tissue was evaluating by western blotting methods.Results:1.Hydroxyl activating derivate cholesterol was obtained according to the reaction of cholesterol with carbonyldiimidazole（CDI）.The cholesterol grafting ratio is about 5%by calculating the integral area of characteristic peaks in¹H NMR spectra.After transforming into aqueous phase,PEI-g-Chol/SPIO nanocomposites show good water dispersibility with a diameter of 61.7±18.5 nm,and positive zeta potential（46.7±0.8 m V）obtained from DLS.A higher T₂relaxivity of the PEI-g-Chol/SPIO(423 Fe m M^-1s^-1)was observed at 1.5T magnetic field.2.PEI-g-Chol/SPIO nanoclusters exhibited high efficiency of siRNA binding efficiency on condition that nitrogen/nucleic acid phosphate（N/P）ratio was fixed at 25.The siRNA sequences targeting human MUC4（sense sequence:5’-GAACAACUUCAGUCCAACUTT-3’）was screened by human Mucin4（MUC4）m RNA sequence.SiRNA could be released from PEI-g-Chol/SPIO/siRNA nanocomposites with heparin sodium/siRNA mass ratios of 0,5,10,20 and 40.Serum stability assay demonstrated that PEI-g-Chol/SPIO/siRNA nanocomposites with satisfactory stability after 48 hours of incubating with50%serum.Phagocytosis assay indicated thatPEI-g-Chol/SPIO/siRNA nanocomposites could be safely engulfed by the cell at an iron concentration of 6ug/m L.Meanwhile,PEI-g-Chol/SPIO nanovectors have significant of siRNA transfection and MUC4 gene silencing efficiencies in vitro.3.In vivo studies have indicated that PEI/SPION/siRNA molecular probe could inhibit the growth of tumor and reduce the expression of MUC4 protein in the tumor after in situ injection of PEI/SPION/siRNA.Conclusion:1.PEI/SPION vector can automatically load and release siRNA and has excellent T₂relaxivity at 1.5 T magnetic field.2.PEI/SPION/siRNA molecular probe was high efficiency of MUC4 gene silencing efficiency through phagocytosis of cancer cells.PEI/SPION/siRNA molecular probe can inhibit the growth of tumor and reduce the expression of MUC4 protein in tumor.3.PEI/SPION vector is quite suitable for acting either as an MR imaging contrast agent or as a medical image visualization genetic vector.4.PEI/SPION/siRNA multifunctional imaging molecular probe has the potential to diagnose and treat pancreatic cancer.