MR Imaging Of Pancreatic Cancer Using Molecular Probe Targeting Survivin Gene

Part ⅠThe preparation of MR Molecular Probe Targeted Survivin Gene In Pancreatic CancerObjective:To construct chitosan modified MNPs(magnetic nanoparticles) and a novel magnetic molecular probe targeting survivin gene over-expressed in pancreatic cancer cells. The characterization of the MNPs was discussed in this section.Materials and methods:1. Preparation of γ-Fe2O3by chemical co-precipi-tation:Firstly, a200mL mixed solution of FeCl3-6H2O (0.01M) and FeSO4-7H2O (0.006M) at pH1.7was prepared under a protection of N2. Then, aqueous ammonia solution (1.5M) was dropped into the mixed solution with violently stirring until the pH of the solution was raised to9to get black magnetite (Fe3O4), then it washed with water5times using magnetic separation technique. PH of the magnetic particles were adjusted to3.0using0.1M HCl. The γ-Fe2O3nanoparticles was obtained by oxidizing the Fe3o4in airstream for1h at about95-100℃until the black color changed to reddish-brown. Secondly, the γ-Fe2O3nanoparticles was modified by chitosan (labeled as CS@MNPs):0.03g chitosan was dissolved in0.5%(v/v) acetic acid solution, then added into solution of γ-Fe2O3(20mL3.8mg/mL) with violently stirring for4h in air. Then the mixture was washed with water using magnetic separation to remove the free chitosan, the CS@MNPs was dispersed in water and its pH was adjusted to5.0using0.1M HC1, then kept in4℃for further use.2. The synthesis of MR Molecular Probe(Survivin targeted magnetic probe, labeled as Sur-MNPs):(1)3.5OD (Optical density, OD) Survivin antisense oligodeoxy-nucleotide, ASODN) was activated by EDC/NHS(1-ethyl-3-(3-dimethyl-amino-propyl)-1-carbo-DiImideh ydrochlori-de/N-hydroxysuccinimide) at25℃for20min before adding into MNPS, and then the mixture was shaken for2h in a swing bed at room temperature. The mixture was washed three cycles to get the probe, ultimately diluted into water.The morphology and core size of CS@MNPs and Sur-MNPs were characterized by TEM (Transmission electron microscopy, TEM). Fourier transform infrared spectroscopy (FTIR) was used to detect the groups on the surface of CS@MNPs. The magnetic property was performed with a Vibrating Sample Magnetometer (VSM) and a MR imager. The stability of our targeted probe was obversed by agarose gel electrophoresis.Results:ASODN of survivin gene was successfully conjugated to the chitosan coated MNPs to give Sur-MNPs.The peak of amino group in Fourier transform infrared spectroscopy and thermogravimetric analysis (TGA) was performed to confirm the conjunction of chitosan. The CS@MNPs and Sur-MNPs had a suitable size (12nm sized core), high stability, and dispersed well in water without aggregation. Relaxation rate of CS@MNPs68.25mM-1×S-1was similar to the value as reported. The result of agarose gel electrophoresis proved that antisense oligodeoxynucleotide was linked to the surface of CS@MNPs to get a novel MR molecular probeConclusions: The molecular probe prepared in this article with a property of good stability, dispersion and optimal magnetic property making a promising for the further experiment in vivo and vitro. Part ⅡThe Pancreatic BxPC-3Cells Cultured With the MR Molecular Probe in VitroObjective:Studying the interactions of pancreatic cells with CS@MNPs and Sur-MNPs whether the probe can be used to transfect the pancreatic cells effectively.Materials and methods: MNPs, CS@MNPs and Sur-MNPs were cultured with the pancreatic cells for24h and then analyzed with series of test as follows:(1)A Prussian blue staining assay was used to research the cellular uptake of nanopar-ticles;(2)MTT (Microculture tetrazolium assay, MTT) assay was utilized as cell viability evaluation to detect the biomaterial toxicity of cells using the healthy lung fibroblast cells as a contrast group.(3) TEM:The labeled cells were washed with water three times and then fixed in1%osmic acid for2h at4℃. Subsequently, the cells were dehydrated with a series of alcohol concentrations, treated with propylene epoxide and finally, embedded with epoxy resin, cut into ultra-thin sections and observed under a TEM.(4)To study the MRI of cells in vitro, the suspensions of labeled cells was were prepared with1%agarose in1.5ml eppendorff tubes and then scanned under a MR imager by using surface coil (repetition time (TR)2500ms, and echo time(TE) from22ms to352ms). Distilled water and the unlabeled cells served as the control. To get T2value of MR it was then executed straight on the imaging using a circular0.32cm2.Results: Many blue particles in cells could be seen in cells incubated with CS@MNPs and MR probe, while the blue particles nearly can’t be seen in cells labeled with γ-Fe2O3. This demonstrates that the targeted probe can transfect the BxPC-3cell line well. The highest uptake amount of the three groups was Sur-MNPs (18.24±0.79pg); the group of γ-Fe2O3was the lowest (9.32±0.21pg).These data of MTT show that after coating with chitosan the nanoparticles manifest lower cytotoxicity than γ-Fe2O3. According to the result of TEM, the nanoparticles were mostly dispersed in cytoplasm and none seen in cytoblast. The mean T2relaxation time of unlabeled cells, cells labeled with MNPs, CS@MNPs, and Sur-MNPs were331.5±18.3ms,219.5±21.8ms,218.0±17.3ms, and171.7±32.5ms respectively.Conclusions:(1) The results proved that CS@MNPs and Sur-MNPs can label cells efficiently. After coating with chitosan, the toxicity of unmodified MNPs t can be reduced.(2) The targeted probe can enter cells and located in cytoplasm, making a reduction in T2signal. So, Sur-MNPs is a good MR molecular probe targeting pancreatic tumors. Part ⅢMR Imaging of Pancreatic Cancer in Animal Models Using MR Targeted ProbeObjective:1.Establish the orthotopic nude mice bearing human pancreatic cancer.2. The efficacy of targeted probe as negative contrast and the its dispersion in mice were evaluate by contrast MRI to Prussian blue staining.Materials and methods:1. the human pancreatic cancer cell line transfected with GFP (green fluorescent protein, GFP) was adherent cultured and then digested with pancreatin, and the suspension of cells was injected subcutaneously. When the tumor formed, part of the tumor was taken and sutured to the pancreas of nude mouse.2. Parameter of MR:T2WI(TR/TE74000ms/71ms; Slice thickness lmm, gap lmm, FOV read41mm.T2map:TR/TE1850/4.9ms, slice thickness1mm, gap1mm, FOV read41mm. Before injected with targeted probe through tail vein (probe0.5mg/ml,200ul) the tumor-bearing mice were scanned precontrast. The enhancement scanning was performed at the following time-point30min,12h,24h and42h. Three mice were sacrificed at each time-point.the normal tissues including liver, spleen, kidney, pancreas and tumor was collected and examined by HE and Prussian blue staining.3. The result of our probe used as MR contrast was detected by analyzing the manifests of MRI combining with Prussian blue staining. The T2value of pancreatic was get by measure the ROI. The statistical differences of tumor T2value was achieved using one-way ANOVA (One-factor analysis of variance, ANOVA) with SPSS18software package (p<0.05was considered as statistically significant difference).Results:After injecting the probe in24h, the result of Prussian blue staining showed that lots of blue particles disbursed in the periphery of pancreatic tumors, in40h, the result of Prussian blue staining showed that blue particles disbursed in the center of pancreatic indicating that the some particles targeted probe can target to the tumor escaping the phagocytosis of reticuloendothelial system (RES). T2WI of pancreatic tumor showed significance lower than that of precontrast (ANOVA, F=20.812, p=0.00). The difference in postcontrat in24h and40h is also significant (p=0.003).Conclusions:The MR molecular probe reported here can target to the pancreatic cancer making a reduction in the T2signal intensity, bombing with the result of Prussian blue staining, demonstrate that the probe Sur-MNPs is a potential specific probe for pancreatic tumors. However, before application in clinical, such questions as how to amplify the signal need to be solved.