Early Diagnosis Of Pancreatic Cancer Using Gold Nanocage With Gd Probe Targeting Survivin Gene

Part I The preparation and characterization of Molecular Probe Targeted Survivin GeneObjective To construct amination of phosphatide polyethylene glycol(NH2-DSPE-PEG)modified Au NC?Gd,combining it with survivin gene to synthesize a novel molecular probe Sur-Au NC?Gd-Cy7 and the characterization of the probe was detected.Materials and methods1.Design of NH2-PEG-Au NC?Gd:1.1 The synthesis of Au nanocage(Au NC): Firstly,30 m L PVP solution(1mg/m L)was dropped into the flask.The solution was heated up to 90°C and then Add 1m L prepared silver nanoparticle solution.One minute later around 6m L solution of HAu Cl4(0.1m M)was added into the flask until the solution had an optical extinction peak at 755 nm as confirmed by UV spectropy.Once cooled to room temperature,the sample was centrifuged and washed with saturated Na Cl solution to remove Ag Cl and with water several times to remove PVP and Na Cl.The solution was transferred to a centrifuge tube and centrifuged for 15 minutes at 8500 rpm for 3 times.Finally add 1: 1 ethanol/water mixture centrifuged,dispersed in 2m L of deionized water.1.2 Synthesis of Au NC?Gd: Diluted hydrochloric acid was added to the aqueous solution of Au NC to adjust its p H value to about 5.Then add 2mg gadolinium(Gd Cl3)and the mixture were shaken for 1h in a swing bed.The supernatant was centrifuged at 8000 rpm/min for 10 min.5 mg of SH-PEG-NH2 was dissolved in carbonate buffer(20 m L)at p H8 and dispersed ultrasonically for 10 min to sufficiently dissolve and disperse the PEG.Then,the supernatant pellet was resuspended by the above procedure,dispersed by ultrasound for 10 min,and added with 0.1% SDS.After incubation at room temperature overnight,SH-PEG-NH2 reacted fully with Au NC.2.Des i gn of Sur-A u NC ?Gd-C y7 : 8.6 nmol Survi vi n ant i se ns e oligodeoxynucleotide(ASODN)was activated by EDC/NHS(1-ethyl-3-(3-dimethylamino-propyl)-1-carbo-Di Imideh ydrochlori-de/N-hydroxysuccinimide)for 30 min and then dissolved in 200 u L dilute hydrochloric acid at p H 5.Add it to PEGylated Au NC solution and add 10 u L(10mg/m L)of Cy7 fluorescent molecule and the mixture was shaken in a swing bed at room temperature overnight.Centrifuge the supernatant to remove unreacted nucleic acid and fluorescent molecules and resuspend it in carbonate buffer with 0.1% SDS.3.The characterization of Au NC?Gd and Sur-Au NC?Gd-Cy7: The morphology of Au NC?Gd was characterized by Transmission electron microscopy(TEM)and the core size of it was displayed on HRTEM.The colloid stability of Au NC?Gd was surveyed for 2 weeks.The hydrodynamic size and zeta potential of Au NC?Gd and Sur-Au NC?Gd-Cy7 were detected.The surface of probe groups was detected by UV absorption,and the imaging performance of the probe was observed by fluorescence,MRI.Results The peak of survivin and Cy7 on the UV absorption spectrum of Au NC?Gd,indicating that the antisense oligonucleotide and fluorescent Cy7 were successfully connected to Au NC?Gd surface.TEM showed that the core size of the targeted probe was about 35.3 nm with uniform hollow,porous morphology and good dispersibility.The measured r1 value of the relaxation curve of Au NC?Gd was 10.51 m M-1s-1and suitable for MR imaging contrast agents.The T1 signal of the mixture increased evidently according to the increasing concentration of Au NC?Gd on MR imaging.The emission peak of the probe was about 780 nm,showing a bright red fluorescence.Conclusion The molecular probe Sur-Au NC?Gd-Cy7 synthesized in this article showed well dispersion,good colloidal stability and proper magnetic property which is proved to be a promising probe for further experiment in vivo and vitro.Part ?The Pancreatic Cancer Cells Cultured With Molecular Probe Targeted Survivin Gene in VitroObjective Studying the interactions of pancreatic cell line PANC-1,Bx PC-3,XPA-1,human lung fibroblast cell line MRC-5 and human pancreatic ductal epithelial cells HTERT-HPNE with Au NC?Gd and Sur-Au NC?Gd-Cy7.We had researched the imaging capabilities and targeting specificity of the probe in vitro.Materials and methods1.TEM: The labeled cells were washed with PBS three times and then fixed with2.5% glutaraldehyde overnight and with 1% osmium tetroxide solution for 1.5 h.Gradient ethanol dehydration and epoxy resin embedding were used to prepare ultrathin sections.The ultrathin section was double dyed with 1% aqueous solution of uranyl acetate and 0.2% lead citrate.The microstructure was observed under transmission electron microscope at 80 k V.2.MTT(Microculture tetrazolium assay,MTT)assay was utilized as cell viability evaluation to detect the biomaterial toxicity of cells using the human pancreatic ductal epithelial cells HTERT-HPNE were used as control group.3.FITC-PI flow cytometry was used to detect the apoptotic effect.The levels of reactive oxygen species(ROS)in cells were detected by Hoechst staining and reactive oxygen species detection.4.MRI imaging in vitro,the suspensions of labeled cells was were prepared with1% agarose in eppendorff tubes and then scanned under a MR imager by using surface coil(repetition time(TR)490 ms,and echo time(TE)from 14 ms).Distilled water and the unlabeled cells served as the control.Results According to the result of TEM,the Sur-Au NC?Gd-Cy7 were mostly dispersed in cytoplasm of pancreatic cancer cells and none seen in normal pancreatic ductal epithelial cells.This demonstrates that the targeted probe can transfect the pancreatic cancer cell line well.These data of MTT showed that the nanoparticles Au NC?Gd represented low cytotoxicity to both Bx PC-3 cell line and HTERT-HPNE cell line.And Sur-Au NC?Gd-Cy7 can cause pancreatic cancer cell survival rate decreased with no significant effect on normal cells.FITC-PI flow cytometry showed that the ratio of apoptotic cells in Sur-Au NC?Gd-Cy7 group was higher than that in Au NC?Gd and Au NC?Gd-Cy7 groups,and no significant change was observed in control group.Hoechst staining showed that the chromatin of Sur-Au NC?Gd-Cy7 group was significantly condensed and apoptotic bodies appeared with nuclear fragmentation.While the chromatin of the normal control group was uniform without concentration,edge set phenomenon,and the nuclei were stained homogeneously,with no apparent bright blue.The number of apoptotic green fluorescence in Sur-Au NC?Gd-Cy7 group was significantly higher than that in control group after staining with the active oxygen detection kit.MRI manifested that the Sur-Au NC?Gd-Cy7 group showed higher T1 signal than the group of Au NC?Gd.Conclusion Sur-Au NC?Gd-Cy7 with low toxicity can enter cells and located in cytoplasm,making a increase in T1 signal.Therefore,the probe Sur-Au NC?Gd-Cy7 may be a good molecular probe specific targeting pancreatic cancer.Part ?Vivo Imaging of Pancreatic Cancer in Animal Models with Molecular Probe Targeted Survivin GeneObjective To investigate the fluorescence,MR imaging and pathological results of targeted molecular probes in orthotopic transplanted pancreatic cancer models and to analyze the distribution of the molecular probes in vivo.Materials and methods1.Establishment of orthotopically implanted human pancreatic cancer in nude mice: The human pancreatic cancer cell line transfected with GFP(green fluorescent protein,GFP)was cultured and then digested with pancreatin,and the suspension of cells was injected subcutaneously to the mice.When the tumor formed,part of the tumor was taken and sutured to the pancreas of nude mouse.2.Hemocompatibility of targeted probe: Approximately 5 m L of blood was drawn from the rat heart and the supernatant was removed by centrifugation after addition of sterile saline.The precipitated red blood cells were washed with sterile saline 2-3 times with sterile saline dubbed 2% suspension.Take 6 clean test tubes for serial numbering,add 6 m L of erythrocyte suspension to each test tube,tubes 1-4 are the test injection reagents(tube 1 was Au NC?Gd,tube 2 was Au NC?Gd-Cy7,Tube3 and 4 was Sur-Au NC?Gd-Cy7),tube 5 was negative control and tube 6 was positive control.After mixing,incubate at room temperature to observe hemolysis and coagulation imaging.3.Fluorescence,MR imaging and HE staining: In vivo NIRF imaging was acquired after intravenous injection of Sur-Au NC?Gd-Cy7 in different time intervals(12h,24 h,48h,72h).The tumor bearing mice were sacrificed at each time point and the organs were imaged.Nine tumor-bearing mice were divided into three groups.Sur-Au NC?Gd-Cy7,Au NC?Gd-Cy7 and Au NC?Gd were injected into the tail vein of each tumor-bearing mice.MR scanning was performed at each time point of12 h,24h,48 h and 72 h after injection.All mice were sacrificed and the tissues including liver,spleen,kidney,pancreas and tumor were collected and examined by HE staining.Results The hemolysis rates of Au NC?Gd-Cy7,Au NC?Gd-Cy7 and Sur-Au NC?Gd-Cy7 were all less than 5%,which accorded with the requirements of biomedical materials on the hemolysis rate,indicating that the probe has good hemocompatibility.Nearinfrared fluorescence imaging showed that after 12 h injection of probe,most of probe fluorescence was located in tumor area,which could overlap with tumor fluorescence.The signal of tumor tissue increased slightly on MR T1 WI,indicating that the targeting probe can specifically target tumor tissue.The liver,spleen and kidney at each time point had no obvious change on the T1 WI signal before and after the injection of probe,and the HE staining at each time point showed no significant change in the cell structure of each organ.Conclusion The molecular probe can target to the pancreatic cancer making a increase in the T1 signal intensity demonstrating that the probe Sur-Au NC?Gd-Cy7 is a potential specific probe for pancreatic tumors.However,before applied to clinical still need further extensive research.